Combination of reversible and irreversible cell labeling for analizing receptor-ligand koff rate

ABSTRACT

The invention relates to a method of determining the dissociation rate constant (k off ) of a receptor molecule R on a target cell using a combination of reversible and irreversible cell labeling. The invention further relates to a cell comprising such a receptor molecule R, wherein the cell has bound to it such a combination of cell labeling. The invention further relates to a kit and an apparatus useful in performing the methods of the invention. The invention further relates to a method of isolation a high-avidity T cell.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention is a continuation of U.S. patent application Ser.No. 16/314,126, filed Dec. 28, 2018, which was filed under 35 U.S.C. §371 as the U.S. national phase of International Application No.PCT/EP2017/065755, filed Jun. 27, 2017, which designated the U.S. andclaims the right of priority of European patent application No.16176537.5, filed with the European Patent Office on Jun. 28, 2016. Theentire disclosures of the above-identified priority applications arehereby fully incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 29, 2022, isnamed SCH-4900-CT_SeqListing.txt and is 4 kilobytes in size.

FIELD OF THE INVENTION

The invention relates to a method of determining the dissociation rateconstant (k_(off)) of a receptor molecule R on a target cell using acombination of reversible and irreversible cell labeling. The inventionfurther relates to a cell comprising such a receptor molecule R, whereinthe cell has bound to it such a combination of cell labeling. Theinvention further relates to a kit and an apparatus useful in performingthe methods of the invention. The invention further relates to a methodof isolation a high-avidity T cell.

BACKGROUND

The importance of cellular adaptive immunity in the control of viral andsome bacterial infections has been convincingly demonstrated bydepletion or adoptive transfer experiments, as well as in genetic mousemodels. Thereby, antigen-specific CD8+ T cells seem to play a dominantrole for protection against intracellular pathogens. Also in certaincancers, the presence of functional CD8+ T cells could be associatedwith the induction of tumor regression. Different immunotherapeuticapproaches therefore aim at the reconstitution of a functional CD8+ Tcell response in patients. If antigen-specific T cells are still presentbut silenced by the micromilieu, application of ‘checkpoint inhibitors’can serve to re-activate the efficacy of existing T cells. However, ifendogenous antigen-specific T cells are lacking or existing cells carryT cell receptors (TCRs) with weak antigen recognition, adoptive T celltransfer of more potent T cells might represent a promising approach torestore a protective antigen specific CD8+ T cell response. Researchover the last decades has indicated that not only the quantity but alsothe quality of prevalent or adoptively transferred T cells correlateswith the success of T cell based immunotherapies. Thus, it is of highinterest in the field to characterize the functional capacity of definedantigen-specific T cell populations for both diagnostic as well astherapeutic applications.

An important parameter to describe T cell functionality is referred toas ‘T cell avidity’, which is commonly assessed on the functional level,e.g. as cytokine production after antigen-specific stimulation. T cellswith high functional avidity could be shown to be superior in clearingviral infections or inducing tumor regression. A major part of T cellavidity is hard-wired within the TCR and can be assessed as structuralavidity, which is the binding strength of the TCR to its cognate ligand,the peptide major histocompatibility complex (pMHC).

Nauerth et al., 2013, Science Translational Medicine, 5(192):192ra87,reported on the so-called ‘TCR-ligand k_(off)-rate assay’, to quantifyan important component of structural TCR avidity. This assay is based onreversible multimers, so called Streptamers. Recombinantly expressedpMHCs can be multimerized on a Streptactin backbone over a shortStreptag sequence, which is fused to the C-term of the pMHCs. Thesemultimeric complexes can stably bind to TCRs expressed on the surface ofliving T cells. With the addition of biotin, however, the multimericcomplex is disrupted, leaving monomeric TCR-pMHC complexes on the T cellsurface. This monomeric binding is not stable and the pMHCs dissociatefrom the TCRs with kinetics depending on the TCR-pMHC binding strength.If the pMHCs are fluorescently labelled, their dissociation can bemonitored by the decay of the fluorescent dye. In contrast to othermethods assessing structural avidity, the TCR-ligand k_(off)-rate assayenables measurement of the dissociation of truly monomeric pMHCs boundto TCRs expressed on the surface of living T cells. Using thek_(off)-rate assay with the help of fluorescence microscopy, theinventors could demonstrate a clear correlation between the TCR-pMHCbinding half-life time (t_(1/2)) and the functionality of correspondingT cells. T cells with a slow t_(1/2) showed not only higher functionalavidity in vitro, but also a higher protective capacity againstinfection in vivo.

Monitoring the fluorescence decay by real-time microscopy offers thepossibility to accurately analyze the TCR-ligand k_(off)-rates on thesingle cell level. Unfortunately, the assay setup is quite labourintensive and requires specific instrumentation. Hebeisen et al. 2015,Cancer Res, 75(10):1983-91 proposed to combine k_(off)-rate measurementswith previous sorting or cloning of analyzed T cells. However, this isnot only a time consuming procedure, it also bears the risk to biasobtained results as in vitro expansions protocols often change thecomposition of complex T cell populations.

It is thus object of the invention to provide means and methods that atleast partially overcomes these drawbacks.

SUMMARY OF THE INVENTION

The present invention relates to a method of determining thedissociation rate constant (k_(off)) of a receptor molecule R on atarget cell and a first receptor binding site B1, comprising detecting afirst detectable label attached to the target cell and a seconddetectable label attached to the target cell, wherein the cell has beencontacted with (i) a first receptor binding reagent, the first receptorbinding reagent comprising at least one first receptor binding site B1,wherein the first receptor binding site B1 specifically binds to saidreceptor molecule R, the first receptor binding reagent furthercomprising at least one first binding partner C1, wherein the firstbinding partner C1 is capable of being reversibly bound to a firstbinding site Z1 of a first multimerization reagent, (ii) a firstmultimerization reagent, the first multimerization reagent comprising atleast two first binding sites Z1 for the reversible binding of the firstbinding partner C1 of the first receptor binding reagent, wherein thefirst receptor binding reagent (i) and the first multimerization reagent(ii) form a first multivalent binding complex that binds to said targetcell, the first multivalent binding complex comprising at least two ofsaid first receptor binding reagents bound to one said firstmultimerization reagent; and wherein said first detectable label isbound or capable of binding to said first receptor binding reagent; andwherein said second detectable label is essentially irreversiblyattached to said receptor molecule R, wherein the first detectable labeland the second detectable label are different from each other.

The invention further encompasses that the cell may have further beencontacted with (iii) a second receptor binding reagent, the secondreceptor binding reagent comprising at least one second receptor bindingsite B2, wherein the second receptor binding site B2 specifically bindsto said receptor molecule R, the second receptor binding reagent furthercomprising at least one second binding partner C2, wherein the secondbinding partner C2 is capable of being stably bound to a second bindingsite Z2 of a second multimerization reagent, and (iv) a secondmultimerization reagent, the second multimerization reagent comprisingat least two second binding sites Z2 for the stable binding of thesecond binding partner C2 of the second receptor binding reagent,wherein the second receptor binding reagent (iii) and the secondmultimerization reagent (iv) form a second multivalent binding complexthat binds to said target cell, the second multivalent binding complexcomprising at least two of second receptor binding reagents bound to onesaid second multimerization reagent; and wherein said second detectablelabel is bound to said second multivalent binding complex.

The invention also encompasses that the cell may have further beencontacted with (iii′) a multimeric receptor binding reagent M2, themultimeric second receptor binding reagent comprising at least twosecond receptor binding sites B2, wherein the second receptor bindingsite B2 specifically binds to said receptor molecule R, wherein themultimeric receptor binding reagent M2 essentially irreversibly binds tosaid target cell via said receptor molecule R, wherein said seconddetectable label is bound to said multimeric receptor binding reagentM2.

The invention also encompasses that the cell may have further beencontacted with (iii″) an irreversible receptor binding reagent I2,wherein the irreversible receptor binding reagent I2 specifically bindsto said receptor molecule R, wherein the irreversible receptor bindingreagent I2 essentially irreversibly binds to said receptor molecule R,wherein said second detectable label is bound to said irreversiblereceptor binding reagent I2.

The invention further encompasses a cell comprising at least threereceptor molecules R, wherein the cell has bound to at least tworeceptor molecules R (i) a first receptor binding reagent, the firstreceptor binding reagent comprising at least one first receptor bindingsite B1, wherein the first receptor binding site B1 specifically bindsto said receptor molecule R, the first receptor binding reagent furthercomprising at least one first binding partner C1, wherein the firstbinding partner C1 is capable of being reversibly bound to a firstbinding site Z1 of a first multimerization reagent, and (ii) a firstmultimerization reagent, the first multimerization reagent comprising atleast two first binding sites Z1 for the reversible binding of the firstbinding partner C1 of the first receptor binding reagent, wherein thefirst receptor binding reagent (i) and the first multimerization reagent(ii) form a first multivalent binding complex that binds to said targetcell, the first multivalent binding complex comprising at least two ofsaid first receptor binding reagents bound to one said firstmultimerization reagent; and wherein a first detectable label is boundto said first receptor binding reagent; and wherein a second detectablelabel is essentially irreversibly attached to at least one receptormolecule R.

The invention further encompasses that the cell may have further boundto at least two receptor molecules R (iii) a second receptor bindingreagent, the second receptor binding reagent comprising at least onesecond receptor binding site B2, wherein the second receptor bindingsite B2 specifically binds to said receptor molecule R, the secondreceptor binding reagent further comprising at least one second bindingpartner C2, wherein the second binding partner C2 is capable of beingstably bound to a second binding site Z2 of a second multimerizationreagent, and (iv) a second multimerization reagent, the secondmultimerization reagent comprising at least two second binding sites Z2for the stable binding of the second binding partner C2 of the secondreceptor binding reagent, wherein the second receptor binding reagent(iii) and the second multimerization reagent (iv) form a secondmultivalent binding complex that binds to said target cell, the secondmultivalent binding complex comprising at least two of second receptorbinding reagents bound to one said second multimerization reagent; andwherein said second detectable label is bound to said second multivalentbinding complex.

The invention also encompasses that the cell may have further bound toat least two receptor molecules R (iii′) a multimeric receptor bindingreagent M2, the multimeric second receptor binding reagent comprising atleast two second receptor binding sites B2, wherein the second receptorbinding site B2 specifically binds to said receptor molecule R, whereinthe multimeric receptor binding reagent M2 essentially irreversiblybinds to said cell via said at least two receptor molecules R, whereinsaid second detectable label is bound to said multimeric receptorbinding reagent M2.

The invention also encompasses that the cell may have further bound to areceptor molecule R (iii″) an irreversible receptor binding reagent I2,wherein the irreversible receptor binding reagent I2 specifically bindsto said receptor molecule R, wherein the irreversible receptor bindingreagent I2 essentially irreversibly binds to said receptor molecule R,wherein said second detectable label is bound to said irreversiblereceptor binding reagent I2

The invention further encompasses a reagent kit for determining thedissociation rate constant (k_(off)) of a receptor molecule R on atarget cell and a first receptor binding site B1, wherein the kitcomprises (i) a first receptor binding reagent, the first receptorbinding reagent comprising at least one first receptor binding site B1,wherein the first receptor binding site B1 specifically binds to saidreceptor molecule R, the first receptor binding reagent furthercomprising at least one first binding partner C1, wherein the firstbinding partner C1 is capable of being reversibly bound to a firstbinding site Z1 of a first multimerization reagent, and (ii) a firstmultimerization reagent, the first multimerization reagent comprising atleast two first binding sites Z1 for the reversible binding of the firstbinding partner C1 of the first receptor binding reagent, wherein thefirst receptor binding reagent (i) and the first multimerization reagent(ii) form a first multivalent binding complex that binds to said targetcell, the first multivalent binding complex comprising at least two ofsaid first receptor binding reagents bound to one said firstmultimerization reagent; wherein said first detectable label is bound orcapable of binding to said first receptor binding reagent.

The kit further comprises (iii) a second receptor binding reagent, thesecond receptor binding reagent comprising at least one second receptorbinding site B2, wherein the second receptor binding site B2specifically binds to said receptor molecule R, the second receptorbinding reagent further comprising at least one second binding partnerC2, wherein the second binding partner C2 is capable of being stablybound to a second binding site Z2 of a second multimerization reagent,and (iv) a second multimerization reagent, the second multimerizationreagent comprising at least two second binding sites Z2 for the stablebinding of the second binding partner C2 of the second receptor bindingreagent, wherein the second receptor binding reagent (iii) and thesecond multimerization reagent (iv) form a second multivalent bindingcomplex that binds to said target cell, the second multivalent bindingcomplex comprising at least two of second receptor binding reagentsbound to one said second multimerization reagent; wherein a seconddetectable label is bound to said second multivalent binding complex; or(iii′) a multimeric receptor binding reagent M2, the multimeric secondreceptor binding reagent comprising at least two second receptor bindingsites B2, wherein the second receptor binding site B2 specifically bindsto said receptor molecule R, wherein the multimeric receptor bindingreagent M2 essentially irreversibly binds to said target cell via saidreceptor molecule R, wherein a second detectable label is bound to saidmultimeric receptor binding reagent M2; or (iii″) an irreversiblereceptor binding reagent I2, wherein the irreversible receptor bindingreagent I2 specifically binds to said receptor molecule R, wherein theirreversible receptor binding reagent I2 essentially irreversibly bindsto said receptor molecule R, wherein a second detectable label is boundto said irreversible receptor binding reagent I2; and wherein the firstdetectable label is not the second detectable label.

The present invention further encompasses an apparatus comprising afirst container containing a target cell comprising a receptor moleculeR, and (i) a first receptor binding reagent, the first receptor bindingreagent comprising at least one first receptor binding site B1, whereinthe first receptor binding site B1 specifically binds to said receptormolecule R, the first receptor binding reagent further comprising atleast one first binding partner C1, wherein the first binding partner C1is capable of being reversibly bound to a first binding site Z1 of afirst multimerization reagent, and (ii) a first multimerization reagent,the first multimerization reagent comprising at least two first bindingsites Z1 for the reversible binding of the first binding partner C1 ofthe first receptor binding reagent, wherein the first receptor bindingreagent (i) and the first multimerization reagent (ii) are capable offorming a first multivalent binding complex that binds to said targetcell, the first multivalent binding complex comprising at least two ofsaid first receptor binding reagents bound to one said firstmultimerization reagent; and wherein said first detectable label isbound or capable of binding to said first receptor binding reagent; andwherein said second detectable label is essentially irreversiblyattached to said receptor molecule R wherein the first detectable labelis not the second detectable label; and a second container containing afluid comprising (a) a competition reagent CR, wherein the competitionreagent CR is capable of competing with first binding partner C1 for thebinding to the first binding site Z1 on the first multimerizationreagent; or (b) a metal chelating reagent, wherein the metal chelatingreagent is preferably EDTA or EGTA, wherein the first container and asecond container are connected such that a fluid can be transferred fromthe second container to the first container.

The first container of the apparatus may further contain (iii) a secondreceptor binding reagent, the second receptor binding reagent comprisingat least one second receptor binding site B2, wherein the secondreceptor binding site B2 specifically binds to said receptor molecule R,the second receptor binding reagent further comprising at least onesecond binding partner C2, wherein the second binding partner C2 iscapable of being stably bound to a second binding site Z2 of a secondmultimerization reagent, and (iv) a second multimerization reagent, thesecond multimerization reagent comprising at least two second bindingsites Z2 for the stably binding of the second binding partner C2 of thesecond receptor binding reagent, wherein the second receptor bindingreagent (iii) and the second multimerization reagent (iv) are capable offorming a second multivalent binding complex that binds to said targetcell, the second multivalent binding complex comprising at least two ofsecond receptor binding reagents bound to one said secondmultimerization reagent; and wherein said second detectable label isbound to said second multivalent binding complex.

The first container of the apparatus may further contain (iii′) amultimeric receptor binding reagent M2, the multimeric second receptorbinding reagent comprising at least two second receptor binding sitesB2, wherein the second receptor binding site B2 specifically binds tosaid receptor molecule R, wherein the multimeric receptor bindingreagent M2 essentially irreversibly binds to said target cell via saidreceptor molecule R, wherein said second detectable label is bound tosaid multimeric receptor binding reagent M2.

The first container of the apparatus may further contain (iii″) anirreversible receptor binding reagent I2, wherein the irreversiblereceptor binding reagent I2 specifically binds to said receptor moleculeR, wherein the irreversible receptor binding reagent I2 essentiallyirreversibly binds to said receptor molecule R, wherein said seconddetectable label is bound to said irreversible receptor binding reagentI2.

The invention further encompasses a method of isolating a high-avidity Tcell comprising (a) determining the dissociation rate constant (k_(off))of a T cell in a sample obtained from a subject using a method of theinvention described herein and (b) isolating said T cell from a sampleobtained from said subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows a schematic depiction of a first multimerization reagent(25) comprising at least two first binding sites Z1 (26) and optionallya third detectable label (93); a second multimerization reagent (35)comprising at least two second binding sites Z2 (36) and a seconddetectable label; a first receptor binding reagent (20) comprising afirst receptor binding site B1 (71), a first detectable label (91), anda first binding partner C1 (81); a second receptor binding reagent (30)comprising a second receptor binding site B2 (72) and a second bindingpartner C2 (82); a multimeric receptor binding reagent M2 (40)comprising at least two second receptor binding sites B2 (72) and asecond detectable label (92); an irreversible receptor binding reagentI2 (50) comprising an irreversible receptor binding site B3 (73) and asecond detectable label (92); and a target cell (10) comprising at leastone receptor molecule R (11).

FIG. 1B illustrates the principle of dissociation rate constant(k_(off)) determination using a first multimerization reagent (25) and asecond multimerization reagent (35). The target cell (10) has bound toat least two receptor molecules R (11) the first receptor binding siteB1 (71) of a first receptor binding reagent (20). The first receptorbinding reagent (20) comprises a first receptor binding site B1 (71), afirst detectable label (91) and a first binding partner C1 (81). Atleast two first receptor binding reagents (20) are reversibly bound to afirst multimerization reagent (25) via the first binding partner C1 (81)comprised in the first receptor binding reagents (20) and the firstbinding site Z1 (26) comprised in the first multimerization reagent(25). The first multimerization reagent (25) optionally comprises athird detectable label (93). Furthermore, the target cell (10) has boundto at least two receptor molecules R (11) the second receptor bindingsite B2 (72) of a second receptor binding reagent (30). At least twosecond receptor binding reagents (30) are stably bound to a secondmultimerization reagent (35) via the second binding partner C1 (82)comprised in the second receptor binding reagents (30) and the secondbinding site Z2 (36) comprised in the second multimerization reagent(35). The second multimerization reagent (35) comprises a seconddetectable label (92). Alternatively, the second detectable label can becomprised in the second receptor binding agent (30) in addition to orinstead of being comprised in the second multimerization reagent (notshown in the Figure). Upon contacting the complex with a competitionreagent CR (60), the competition reagent CR (60) competes with the firstbinding partner C1 (81) comprised in the first receptor binding reagent(20) for the first binding site Z1 (26) comprised in the firstmultimerization reagent (25). Due to binding of the competition reagentCR (60) to the first binding site Z1 (26) comprised in the firstmultimerization reagent (25), the binding between the first receptorbinding reagent (20) and the first multimerization reagent (25) isdisrupted and the first multimerization reagent (25), and eventually thefirst receptor binding reagent (20), detach from the target cell (10).

FIG. 1C shows the principle of dissociation rate constant (k_(off))determination using a first multimerization reagent (25) and amultimeric receptor binding reagent M2 (40). The target cell (10) hasbound to at least two receptor molecules R (11) the first receptorbinding site B1 (71) of a first receptor binding reagent (20). The firstreceptor binding reagent (20) comprises a first receptor binding site B1(71), a first detectable label (91) and a first binding partner C1 (81).At least two first receptor binding reagents (20) are reversibly boundto a first multimerization reagent (25) via the first binding partner C1(81) comprised in the first receptor binding reagents (20) and the firstbinding site Z1 (26) comprised in the first multimerization reagent(25). The first multimerization reagent (25) optionally comprises athird detectable label (93). Furthermore, the target cell (10) has boundto at least two receptor molecules R (11) the second receptor bindingsite B2 (72) of a multimeric receptor binding reagent M2 (40). Themultimeric receptor binding reagent M2 (40) comprises a seconddetectable label (92). Upon contacting the complex with a competitionreagent CR (60), the competition reagent CR (60) competes with the firstbinding partner C1 (81) comprised in the first receptor binding reagent(20) for the first binding site Z1 (26) comprised in the firstmultimerization reagent (25). Due to binding of the competition reagentCR (60) to the first binding site Z1 (26) comprised in the firstmultimerization reagent (25), the binding between the first receptorbinding reagent (20) and the first multimerization reagent (25) isdisrupted and the first multimerization reagent (25), and eventually thefirst receptor binding reagent (20), detach from the target cell (10).

FIG. 1D shows the principle of dissociation rate constant (k_(off))determination using a first multimerization reagent (25) and anirreversible receptor binding reagent I2 (50). The target cell (10) hasbound to at least two receptor molecules R (11) the first receptorbinding site B1 (71) of a first receptor binding reagent (20). The firstreceptor binding reagent (20) comprises a first receptor binding site B1(71), a first detectable label (91) and a first binding partner C1 (81).At least two first receptor binding reagents (20) are reversibly boundto a first multimerization reagent (25) via the first binding partner C1(81) comprised in the first receptor binding reagents (20) and the firstbinding site Z1 (26) comprised in the first multimerization reagent(25). The first multimerization reagent (25) optionally comprises athird detectable label (93). Furthermore, the target cell (10) has boundto a receptor molecule R (11) the irreversible receptor binding reagentI2 (50). The irreversible receptor binding reagent I2 (50) comprises asecond detectable label (92). Upon contacting the complex with acompetition reagent CR (60), the competition reagent CR (60) competeswith the first binding partner C1 (81) comprised in the first receptorbinding reagent (20) for the first binding site Z1 (26) comprised in thefirst multimerization reagent (25). Due to binding of the competitionreagent CR (60) to the first binding site Z1 (26) comprised in the firstmultimerization reagent (25), the binding between the first receptorbinding reagent (20) and the first multimerization reagent (25) isdisrupted and the first multimerization reagent (25), and eventually thefirst receptor binding reagent (20), detach from the target cell (10).

FIGS. 2A-2E: Setup and analysis of the flow cytometry based k_(off)-rateassay

FIG. 2A is a schematic depiction of an examplary setup on a flowcytometer. FIG. 2B is an exemplary depiction of a sample temperaturecontroller with FACS tube for the k_(off)-rate assay. FIG. 2C shows anexemplary gating strategy of the flow based k_(off)-rate assay with Tcell clones: T cell clones expressing the 2C TCR were stained withreversible multimers (comprising Streptactin APC and SIYRYYGL (SEQ IDNO: 10)-H2Kb-Alexa488) and Propidium Iodide (PI) for live/deaddiscrimination. Stained cell were analyzed on a Cyan Lx, 2 mM D-biotinwas added after 30 s and dissociation was observed for 10 min. Sampleswere gated on living lymphocytes and dissociation of Streptactin-APC andMHC-Alexa488 from these cells were observed. FIG. 2D shows a reductionof data to visualize dissociation kinetics: APC and Alexa488Fluorescence intensity of living lymphocytes was plotted over thecomputed time of analysis in FlowJo. The computed time was divided in200 gates, and mean fluorescence intensity (MFI) of all cells containedin one gate was calculated. MFI values of APC (left axis) and Alexa488(right axis) were plotted over time of analysis in Graph Pad Prism (topgraph). Data points for analysis were selected and fitted with anexponential decay (bottom graph). FIG. 2E is a direct analysis ofdissociation kinetics of all cells: Fluorescence values of all livinglymphocytes are directly exported to Graph Pad Prism. Data points foranalysis are selected and are fitted with an exponential decay.

FIGS. 3A-3D: Flow cytometry based koff-rate assay with CMV-specific Tcell clones

FIG. 3A to FIG. 3C show k_(off)-rate assays of human CMV-specific T cellclones: T cell clones were stained with specific reversible multimers(comprising Streptactin APC and MHC-Alexa488) and Propidium Iodide (PI)for live/dead discrimination. K_(off)-rate assay was performed on a CyanLx. Fluorescence values of Alexa 488 and APC staining of living cellsare plotted over the time of analysis, data points for analysis areselected as described and fitted with an exponential decay. FIG. 3Dshows a comparison of the TCR-pMHC binding half-life times assessed onthe microscope and on the flow cytometer.

FIGS. 4A-B: Principle of double staining with reversible andnon-reversible multimers

FIG. 4A is a schematic representation of the staining of a CD8+ T cellwith a reversible and a non-reversible multimer (top left) and thedissociation of the reversible multimer after the addition of D-biotin(bottom right). FIG. 4B shows that T cells derived from HumanCMV-specific T cell clones were stained with specific reversiblemultimer (comprising Streptactin APC and HLA-B8 1E188-96-Alexa488),Propidium Iodide (PI) for live/dead discrimination and with thespecific, non-reversible multimer (comprising Streptavidin BV421 andunlabeled B8 1E188-96 molecules). Dot plot of the double staining(Streptactin APC and Streptavidin BV421, gated on living, CD8+lymphocytes) of a human CMV-specific T cell clone before addition ofD-biotin (left). Staining intensities of the backbone of thenon-reversible multimer (Streptavidin BV421) and of the backbone(Streptactin APC) and the pMHC molecules (Alexa488) of the reversiblemultimer over time of analysis with addition of D-biotin after 30 s.

FIGS. 5A-F: Reversible multimer staining intensity and k_(off)-rateafter double staining

FIG. 5A depicts titration of incubation time and concentration of thenon-reversible multimer staining: PBMCs from a CMV positive (B7 pp65)donor were incubated with the specific reversible multimer (comprisingStreptactin APC and HLA-B7 pp65-Alexa488), antibodies against CD8 PE-Cyand Propidium Iodide (PI) for live/dead discrimination. After washing,samples were incubated with the specific non-reversible multimer, withincubation time and dilution of the multimer indicated above the dotplots (see also Material and Methods). Dot plots show CD8 Pe-Cy7 andStreptavidin BV421 staining of CD8+ living lymphocytes. FIG. 5B and FIG.5C show histograms of the staining of the non-reversible multimer(Streptavidin BV421) and the reversible multimer (MHC Alexa 488 andStreptactin APC) of the gates depicted in FIG. 5A). FIG. 5D showsTCR-pMHC k_(off)-rates of B7pp65 specific T cells contained in thesamples described in A). FIG. 5E and FIG. 5F shows the results of twoCMV-specific T cell clones (specific for HLA B8 1E188-96) that wereeither stained with the reversible multimer alone or in combination withthe non-reversible multimer conjugated with BV421 or with PE. Allsamples were stained with Propidium Iodide (PI) for live/deaddiscrimination. K_(off)-rates were measured from living lymphocytes.

FIGS. 6A-D: Double staining and sort of B7 pp65 specific T cellpopulation

PBMCs from a CMV positive donor (specific for HLA B7 pp65) were stainedwith the specific reversible multimer (comprising Streptactin APC andMHC-Alexa488), antibodies against CD8 and Propidium Iodide (PI) forlive/dead discrimination. Cells were split in two samples. FACS sortingfor living CD8+ Streptactin APC+ lymphocytes was performed on a MoFlo(Beckman Coulter) with one sample. The other sample was additionallystained with the non-reversible multimer (comprising Streptavidin PE).FIG. 6A shows dot plots of the Streptavidin PE and the CD8 eF450staining of living lymphocytes (left picture) and the MHC Alexa488 andStreptactin staining of Streptavidin+ cells of the double stained sample(right picture). FIG. 6B shows k_(off)-rate assays with reversiblemultimer stained B7pp65 specific T cells while FIG. 6C showsk_(off)-rate assays with double stained B7pp65 specific T cells.Fluorescence values of Alexa 488 and APC staining of the analyzed cellsare plotted over the time of analysis, data points for analysis areselected as described and fitted with an exponential decay. FIG. 6Dshows a comparison of the TCR-pMHC binding half-life times of the sortedand the double stained sample.

FIG. 7: Ex vivo k_(off) rate measurement of an oligo clonal HLA*0702/CMVpp65 specific CD8 T cell population.

PBMCs were isolated by Ficoll gradient centrifugation from fresh bloodof a healthy donor. Boolean gating on single living CD19− CD8+non-reversible pMHC-PE+ T cells. Data of Streptactin APC and reversiblepMHC-Alexa488 over time were exported to GraphPad PRISM to fit anexponential decay curve to determine the k_(off) rate.

FIG. 8: Ex vivo k_(off) rate measurement of an oligo clonal HLA*0201/CMVpp65 specific CD8 T cell population (donor HZ961).

k_(off) rate measurement of cryopreserved PBMCs isolated by Ficollgradient centrifugation from fresh blood of a healthy donor. Booleangating on single living CD19− CD8+ non-reversible pMHC-PE+ T cells. Dataof Streptactin APC and reversible pMHC-Alexa488 over time were exportedto GraphPad PRISM to fit an exponential decay curve to determine thek_(off) rate.

FIG. 9: Ex vivo k_(off) rate measurement of an oligo clonal HLA*0201/CMVpp65 specific CD8 T cell population (donor HZ510).

k_(off) rate measurement of cryopreserved PBMCs isolated by Ficollgradient centrifugation from fresh blood of a healthy donor. Booleangating on single living CD19− CD8+ non-reversible pMHC-PE+ T cells. Dataof Streptactin APC and reversible pMHC-Alexa488 over time were exportedto GraphPad PRISM to fit an exponential decay curve to determine thek_(off) rate.

FIG. 10: Functional characterization of isolated TCRs using k_(off) ratemeasurement on CD8+ J76 tumor cells.

Two HLA*02 01/CMVpp65 specific TCRs were isolated using single clonePCR.

To analyze their structural avidity, TCRs were transduced into CD8+J76tumor cells lacking an endogenous TCR. Boolean gating on single livingCD8+ non-reversible pMHC-PE+ T cells. Data of Streptactin APC andreversible pMHC-Alexa488 over time were exported to GraphPad PRISM tofit an exponential decay curve to determine the k_(off) rate.

FIG. 11: k_(off) rate measurement in a murine model system for high andlow avidity TCR pMHC interaction.

Row a.; SIIQFEKL:H2 kb (SEQ ID NO:14), row b.: SIYNFEKL:H2 kb (SEQ IDNO: 15); row c.: SIINFEKL:H2 kb (SEQ ID NO: 15).

DETAILED DESCRIPTION

For some applications, e.g. the measurement of homogenous populationslike T cell clones or transduced T cells, single cell resolution is notnecessary and a method allowing fast screening of k_(off)-rate valuesusing commonly available instrumentation would be desirable. Theinventors of the present application therefore aimed to transfer theprinciple of the reversible multimer based k_(off)-rate assay toapplications guided by conventional flow cytometry. The inventors havesurprisingly demonstrated that k_(off)-rate kinetics can indeed beacquired with high sensitivity by flow cytometry; thereby, the proceduregenerates highly reliable dissociation measurements with valuescomparable to a microscopy guided assay. The inventors have furthersurprisingly demonstrated that this procedure may be transferred to theanalysis of antigen-specific T cell populations directly ex vivo. Theseresults could not have been expected before, because in vivo antigenspecific T cell populations are often quite small and the presence ofadditional (unspecific) cells limits the tracking of staining kinetics,like it is necessary for k_(off)-rate measurements. Hebeisen et al.2015, Cancer Res, 75(10):1983-91 proposed to combine k_(off)-ratemeasurements with previous sorting or cloning of analyzed T cells.However, the present inventors have recognized that this is not only atime consuming procedure, it also bears the risk to bias obtainedresults as in vitro expansions protocols often change the composition ofcomplex T cell populations. Therefore, the inventors envisioned a directassessment of k_(off)-rate values without cell sorting and in vitro cellexpansion by double staining of antigen-specific T cells with reversibleand non-reversible specific MHC multimers. Staining with non-reversiblemultimers allows to stably identify the antigen-specific targetpopulation while observing the dissociation of the reversible multimers.Importantly, the inventors of the present application were able todemonstrate that the dissociation kinetics of the pMHCs are notinfluenced by additional staining with non-reversible multimers. Hence,multimer double staining in combination with flow cytometry-guidedTCR-ligand k_(off)-rate measurement can be used for analyzing even verysmall antigen-specific T cell populations directly ex vivo.

The present invention therefore encompasses a method of determining thedissociation rate constant (k_(off)) of a receptor molecule R on atarget cell and a first receptor binding site B1, comprising detecting afirst detectable label attached to the target cell and a seconddetectable label attached to the target cell. While the first detectablelabel may be reversibly bound to the cell, the second detectable labelmay be essentially irreversibly bound to the cell. For this purpose, thecell has been contacted with (i) a first receptor binding reagent, thefirst receptor binding reagent comprising at least one first receptorbinding site B1, wherein the first receptor binding site B1 specificallybinds to said receptor molecule R, the first receptor binding reagentfurther comprising at least one first binding partner C1, wherein thefirst binding partner C1 is capable of being reversibly bound to a firstbinding site Z1 of a first multimerization reagent, and a firstmultimerization reagent, the first multimerization reagent comprising atleast two first binding sites Z1 for the reversible binding of the firstbinding partner C1 of the first receptor binding reagent. The firstmultimerization reagent preferably comprises at least 3, preferably atleast 3, preferably 4-20, preferably 4-8, preferably 4 first bindingsites Z1. The first receptor binding reagent (i) and the firstmultimerization reagent (ii) form a first multivalent binding complexthat binds to said target cell, the first multivalent binding complexcomprising at least two of said first receptor binding reagents bound toone said first multimerization reagent. The first multivalent complexpreferably comprises at least 3, preferably at least 4, preferably 4-20,preferably 4-8, preferably 4 first receptor binding reagents bound toone said first multimerization reagent. It is understood that the firstdetectable label is bound or capable of binding to said first receptorbinding reagent. It is further understood that said second detectablelabel is essentially irreversibly attached to said receptor molecule R.It is also understood that the first detectable label and the seconddetectable label are different from each other, meaning that they arenot the same compound. The first multimerization reagent optionallyfurther comprises a third detectable label. It is understood that eachof the first, second, and third detectable label are preferablydifferent from each other and can preferably be distinguished from eachother.

In this context, it is noted that the formation of a complex (C) betweena receptor binding reagent B and its receptor R, e.g. a cell surfacereceptor molecule can be described by a two-state process noted

C

B+R

The corresponding dissociation K_(d) constant is defined as

$K_{d} = \frac{\lbrack B\rbrack + \lbrack R\rbrack}{\lbrack C\rbrack}$

wherein [B], [R], and [C] are the equilibrium molar concentrations ofthe receptor, the receptor binding reagent (ligand) and the respectivecomplex at a given temperature and pressure. The dissociation K_(d)constant can also be expressed as the ratio of the constant of theon-rate (k_(on)) for the speed of association/formation (also calledassociation rate constant) of the complex and the constant of theoff-rate (k_(off)) for the dissociation of the complex (also calleddissociation rate constant) with

$K_{d} = \frac{k_{off}}{k_{on}}$

In the present application, the values of the thermodynamic and kineticconstants K_(d), k_(on) and k_(off) refer to their determination at atemperature of 4° C. and atmospheric pressure of 1.013 bar.

As used herein, “reversible”, when used in the context of a monovalentbinding complex, may be expressed in terms of the k_(off) rate for thebinding between two binding partners, e.g. the binding between areceptor molecule R and its binding partner B. The k_(off) rate forreversible binding may be about 0.5×10⁻⁴ sec⁻¹ or greater, about 1×10⁻⁴sec⁻¹ or greater, about 2×10⁻⁴ sec⁻¹ or greater, about 3×10⁻⁴ sec⁻¹ orgreater, about 4×10⁻⁴ sec⁻¹ of greater, about 5×10⁻⁴ sec⁻¹ or greater,about 1×10⁻³ sec⁻¹ or greater, about 1.5×10⁻³ sec⁻¹ or greater, about2×10⁻³ sec⁻¹ or greater, about 3×10⁻³ sec⁻¹ or greater, about 4×10⁻³sec⁻¹, about 5×10⁻³ sec⁻¹ or greater, about 1×10⁻² sec⁻¹ or greater, orabout 5×10⁻¹ sec⁻¹ or greater. The respective K_(D) value of such areversible binding complex may be in the range of about 1×10⁻¹⁰ M orgreater, about 1×10⁻⁹ M or greater, about 1×10⁻⁸M or greater, about1×10⁻⁷M or greater, about 1×10⁻⁶M or greater, about 1×10⁻⁵ M or greater,about 1×10⁻⁴ M or greater, about 1×10⁻³ M or greater. In contrastthereto, “irreversible” or “essentially irreversible”, which is usedsynonymously and interchangeable, may also be expressed in terms of ak_(off) rate. The k_(off) rate for an (essentially) irreversiblebinding, e.g. between a receptor molecule R and its binding partner B,may be about 1×10⁻⁵ sec⁻¹ or lower, about 1×10⁻⁶ sec⁻¹ or lower, about1×10⁻⁷ sec⁻¹ or lower, about 1×10⁻⁸ sec⁻¹ or lower, about 1×10⁻⁹ sec⁻¹or lower, about 1×10⁻¹⁰ sec⁻¹ or lower. The respective K_(D) value ofsuch a irreversible binding complex may be in the range of about 2×10⁻¹⁰M or less, about 1×10⁻¹¹M or less, or about 1×10⁻¹²M or less, about1×10⁻¹³M or less, or about 1×10⁻¹⁴ M or less. It may be in the range of2×10⁻¹⁰ M to about 10⁻¹⁵ M. The term “about” when used herein inrelation to the k_(off) rate, the k_(on) rate or the K_(d) is meant toinclude an error margin of ±0.1%, ±0.2%, ±0.3%, ±0.4%, ±0.5%, ±0.7±0.9,%±1.0, %, ±1.2%, ±1.4%, ±1.6%, ±1.8%, ±2.0%, ±2.2%, ±2,4,%, ±2.6%,±2.8%, ±3.0%, ±3.5%, ±4.0.%, ±4.5%, ±5.0%, ±6.0%, ±7.0%±, 8.0%, ±9.0%±,10.0%, ±15.0%, or ±20.0%. However, since a receptor molecule R may betypically present in two or more copies on the surface of a target cell,an avidity effect may have to be considered if a reagent that binds tothe cell comprises two or more binding sites B. In such a case, althoughthe binding between a single receptor R and a single binding site B maybe reversible, the binding of a cell comprising multiple R and a reagentcomprising multiple binding sites B may be essentially irreversible. Forsuch a multivalent binding, an apparent k_(off)-value may be defined,wherein the apparent k_(off) value is the k_(off) value that mayapparently be measured, if assumed that the binding is monovalent. Asdiscussed, due to the avidity effect, binding of a cell comprising twoor more receptors R and a reagent, e.g. a multimeric reagent, comprisingmore than one binding sites B may be essentially irreversible, e.g. theapparent k_(off) value may be in the range as defined for anirreversible binding. However, it is possible to reversibly multimerizemultiple binding reagents that each comprise a binding site B. For thispurpose, a multimerization reagent may be used that comprise at leasttwo binding sites Z. The binding reagent comprises, further to thebinding site B, a binding partner C, which may bind to the binding siteZ on the multimerization reagent. Here, the binding between C and Z isreversible, and it can preferably be disrupted, e.g. by adding reagentsthat compete with C for the binding site Z and may displace C from theC:Z complex. Hence, a reversible multimer that has two or more bindingreagents bound to it, each comprising a binding site B, may bind to acell comprising two or more receptors R with a high avidity and anapparent k_(off) value which would normally indicate essentiallyirreversible binding as long as it is in the form of a multimer. Thebinding to the cell may still be reversible, if the multimerizationitself can be reversed as described herein and the monovalent binding ofthe binding site B with the receptor R is reversible. Such reversiblebinding by a reversible multimer is e.g. described in U.S. Pat. No.7,776,562, International Patent application WO 02/054065, orInternational Patent application WO 2013/011011.

As used herein, “detectable label” refers to detectable entities thatcan be used for the detection of a stained cell in flow cytometry.Preferably, the label does not negatively affect the characteristics ofthe cells to be stained or isolated. Examples of labels are fluorescentlabels such as phycoerythrin, allophycocyanin (APC), Brilliant Violet421, Alexa Fluor 488, coumarin or rhodamines to name only a few. Thelabel may be bound to the receptor binding reagent and/or themultimerization reagent. When applicable, a first detectable label ispreferably bound to a receptor binding reagent, while an optional thirddetectable label is preferably bound to a multimerization reagent. Thelabel may be a direct label, i.e. a label bound to one of the members ofthe multivalent binding complex as specified above. In such a case, thelabel might, for example, be covalently coupled (conjugated) to eitherthe receptor binding reagent or the multimerization reagent.Alternatively, the label may be an indirect label, i.e. a label which isbound to a further reagent which in turn is capable of binding to one ofthe members of the multivalent binding complex as specified above. Sucha label may be added before, during or after the multivalent bindingcomplex has been formed. An example for such an indirect label is abis-, tris-, or tetrakis-NTA containing fluorescent dye as described byLata et al., J. Am. Chem. Soc. 2005, 127, 10205-10215 or Huang et al.,Bioconjugate Chem. 2006, 17, 1592-1600. Said label is capable of bindingnon-covalently (via metal chelation) to an oligohistidine tag. Thus, inthis example, the receptor binding reagent and/or the multimerizationreagent may carry an oligohistidine tag (for example, a Fab fragment asreceptor binding reagent that has an oligohistidine tag such as ahexa-histidine tag fused to the C-terminus of the CH1 or the CL-domainor a streptavidin mutein such as the commercially availableStrep-Tactin® mutein (IBA GmbH, Gottingen, Germany) as multimerizationreagent that has an oligohistidine tag fused to the N- or C-terminus ofone of its subunits can be used), thereby being enabled tonon-covalently bind such a NTA based fluorescent dye compound describedby Lata et al, supra or Huang et al, supra. Such a non-covalentlybinding label needs not necessarily be bound to its target (receptorbinding reagent and/or the multimerization reagent) before themultivalent binding complex is formed but can also be added to thesample when the multivalent binding complex forms or after themultivalent binding complex has been formed. Such a non-covalentlybinding label can also be added after the multivalent binding complex isbound to the target cells. Instead of the NTA comprising fluorescentdye:oligohistidine tag binding pair described above, also any otherspecific binding pair such as, for example, digoxigenin and an antidigoxigenin antibody or antibody fragment carrying the fluorescent orother label can be used for indirect labeling. In such a case, thereceptor binding reagent and/or the multimerization reagent isconjugated to/coupled with digoxigenin and an anti digoxigenin antibodyor antibody fragment carrying the chosen label binds (via digoxigenin)to the multimerization reagent or the receptor binding reagent. Theinvention encompasses that the first detectable label or the seconddetectable label may both be a fluorescent dye. An optional thirddetectable label may also be a fluorescent dye. It is preferred that thefirst detectable label is a first fluorescent dye and the seconddetectable label is a second fluorescent dye, wherein the emissionsignal of the first fluorescent dye can preferably be distinguished fromthe emission signal of the second fluorescent dye. An optional thirddetectable label may also be a fluorescent dye that can preferably bedistinguished from both the first and the second fluorescent dye. As anillustrative example, the first detectable label may be Alexa Fluor 488(Alexa488), while the second detectable label may be Brilliant Violet421 (BV421). The optional third detectable label may be allophycocyanin(APC).

As discussed, the target cell has a second detectable label attached toit, wherein the second detectable label is essentially irreversiblyattached to the receptor molecule R. It is understood that there aremultiple possibilities for essentially irreversibly attaching such saidsecond label to a one or more receptor molecule(s) R. The invention thusenvisions, as one possibility, that the second detectable label isattached to the cell via an irreversible multimer. For this purpose, thecell may have further been contacted with (iii) a second receptorbinding reagent, the second receptor binding reagent comprising at leastone second receptor binding site B2, wherein the second receptor bindingsite B2 specifically binds to said receptor molecule R, the secondreceptor binding reagent further comprising at least one second bindingpartner C2, wherein the second binding partner C2 is capable of beingstably bound to a second binding site Z2 of a second multimerizationreagent, and (iv) a second multimerization reagent, the secondmultimerization reagent comprising at least two second binding sites Z2for the stable binding of the second binding partner C2 of the secondreceptor binding reagent. The second multimerization reagent preferablycomprises at least 3, preferably at least 3, preferably 4-20, preferably4-8, preferably 4 second binding sites Z2. The stable binding betweenthe second binding partner C2 and the second binding site Z2 may be anessentially irreversible binding as defined herein. Hence, thedissociation rate constant (k_(off)) for the binding between said secondbinding partner C2 and said second binding sites Z2 can, for example, bein the range as defined herein for an essentially irreversible binding.Here, the second receptor binding reagent (iii) and the secondmultimerization reagent (iv) form a second multivalent binding complexthat binds to said target cell, the second multivalent binding complexcomprising at least two of second receptor binding reagents bound to onesaid second multimerization reagent, and said second detectable label isbound to said second multivalent binding complex. Alternatively, thebinding between the second binding partner C2 and the second bindingsite Z2 may be a reversible binding, and the second binding partner C2and the second binding site Z2 may be any pair of compounds that aredefined for C1 and Z1, with the proviso that the bond between C2 and Z2cannot disrupted by the same method, by which the bond between C1 and Z1can be disrupted. As an illustrative example, C1 may be a streptavidinbinding affinity peptide and Z1 may be streptavidin mutein such asstreptactin, while C2 may be an oligohistidine tag and Z2 is a compoundthat comprises a divalent cation. If biotin is added to these complexes,the bond between C1 and Z1 will be disrupted, while the bond between C2and Z2 will stay intact. Hence, although the bond C2 and Z2 cangenerally be disrupted (e.g. by contacting the complex with ametal-chelating agent, such as EDTA), it can still serve as anirreversible multimer in the methods of the invention, as long as thebond between C2 and Z2 is not disrupted by the same means or methods, bywhich the bond between C1 and Z1 can be disrupted. Consequently, thebinding between Z2 and C2 is preferably irreversible, or preferably notdisruptable by the means that may disrupt the binding between Z1 and C1.The second multivalent complex preferably comprises at least 3,preferably at least 4, preferably 4-20, preferably 4-8, preferably 4second receptor binding reagents bound to one said secondmultimerization reagent. Hence, it is preferably possible to selectivelydisrupt the binding between Z1 and C1 while the binding between Z2 andC2 stays intact. Hence, it may be possible to selectively reverse theattachment of the first detectable label to the cell, while the seconddetectable label will remain attached to the cell. It is furtherunderstood that B1 and B2 may be the same or different. It is preferredthat B1 and B2 are the same.

Another possibility for essentially irreversibly attaching a seconddetectable label to the target cell may be using a binding reagent thatcomprises multiple B2 binding sites. Hence, it is encompassed that themethod of the invention may comprise that the cell has further beencontacted with (iii′) a multimeric receptor binding reagent M2, whereinthe multimeric second receptor binding reagent comprising at least twosecond receptor binding sites B2, preferably at least 3, preferably atleast 4, preferably 4-20, preferably 4-8, preferably 4 receptor bindingsites B2, wherein the second receptor binding site B2 specifically bindsto said receptor molecule R, wherein the multimeric receptor bindingreagent M2 essentially irreversibly binds to said target cell via saidreceptor molecule R, and wherein said second detectable label is boundto said multimeric receptor binding reagent M2. Here the apparentdissociation rate constant (k_(off)) for the binding between saidmultimeric receptor binding reagent M2 and said target cell can, forexample, be about 1×10⁻⁵ sec⁻¹ or lower, about 1×10⁻⁶ sec⁻¹ or lower,about 1×10⁻⁷ sec⁻¹ or lower, about 1×10⁻⁸ sec⁻¹ or lower, about 1×10⁻⁹sec⁻¹ or lower, about 1×10⁻¹⁰ sec⁻¹ or lower. Here again, it isunderstood that B1 and B2 may be the same or different, preferably thesame.

Essentially irreversibly attaching a second detectable label to thetarget cell may also be achieved by using an agent that irreversiblybinds to a receptor R. The invention thus encompasses that the method ofthe invention may comprise that the cell has further been contacted with(iii″) an irreversible receptor binding reagent I2, wherein theirreversible receptor binding reagent I2 specifically binds to saidreceptor molecule R, wherein the irreversible receptor binding reagentI2 essentially irreversibly binds to said receptor molecule R, andwherein said second detectable label is bound to said irreversiblereceptor binding reagent I2. It is understood, that for binding to R, 12may comprise an irreversible binding site B3, that essentiallyirreversibly binds to R and that is preferably different from B1. Herethe dissociation rate constant (k_(off)) for the binding between saidirreversible binding site B3 and said receptor molecule R can, forexample, be about 1×10⁻⁵ sec⁻¹ or lower, about 1×10⁻⁶ sec⁻¹ or lower,about 1×10⁻⁷ sec⁻¹ or lower, about 1×10⁻⁸ sec⁻¹ or lower, about 1×10⁻⁹sec⁻¹ or lower, about 1×10⁻¹⁰ sec⁻¹ or lower.

In the method of the invention, virtually any said target cell can beused that has at least one common receptor molecule that can be used fork_(off) rate measurement. In order to achieve an avidity effect, thereceptor molecule is typically present in two or more copies on thesurface of the target cell. In typical embodiments the target cell is aeukaryotic or prokaryotic cell, preferably a mammalian cell. Themammalian cell may be a lymphocyte or a stem cell. Hence, the targetcell may be a T cell, a T helper cell, a B cell or a natural killercell, such as a CMV-specific a CMV-specific CD8+ T-lymphocyte, acytotoxic T-cell a, memory T-cell and a regulatory T-cell. Likewise, theat least one common (specific) receptor which defines the target cellpopulation may be any receptor for which a k_(off) rate of the bindingto a given binding site B1 can be determined. For example, the receptormay be a receptor defining a population or subpopulation of immunecells, e.g. a population or subpopulation of T cells, T helper cells,for example, CD4+ T-helper cells, B cells or natural killer cells.Examples of T cells include cells such as CMV-specific CD8+T-lymphocytes, cytotoxic T cells, memory T cells and regulatory T cells(Treg). The receptor molecule R may be any receptor present on thetarget cell. However, it is preferred that the receptor is anantigen-specific receptor, such as e.g. a T cell receptor or a B cellreceptor. The receptor may preferably be a T cell receptor while thetarget cell may preferably be a CD8+ T cell. In this context, it isnoted that the term “target cells” as used herein encompasses allbiological entities/vesicles in which a membrane (which can also be alipid bilayer) separates the interior from the outside environment andwhich comprise specific receptor molecules on the surface of thebiological entity. Examples of such entities include, but are notlimited to, a cell, a virus, a liposome, an organelle such asmitochondria, chloroplasts, a cell nucleus or a lysosome.

In the methods of the invention, the first receptor binding site B1 ofthe receptor binding reagent which specifically binds to said receptormolecule R can for example be an MHC molecule. The use of MHC moleculesas first receptor binding site B1 allow the characterization of ak_(off) rate of a T cell receptor of an antigen-specific subpopulationof T cells directly ex vivo. It is noted that the term “MHC molecule”includes MHC molecules that are conjugated with a peptide. The firstreceptor binding site B1 may also be an antibody or a divalent antibodyfragment such as an (Fab)₂′-fragment, divalent single-chain Fv fragment.It might also be a bivalent proteinaceous artificial binding moleculesuch as a dimeric lipocalin mutein that is also known as “duocalin”. Inother embodiments the receptor binding reagent may have a single bindingsite B1, i.e., may be monovalent. Examples of monovalent receptorbinding reagents include, but are not limited to, a monovalent antibodyfragment or a proteinaceous binding molecule with antibody-like bindingproperties, such as a lipocalin mutein.

As discussed herein, the bond between the first binding partner C1 andthe first binding site Z1 should be reversible, i.e. the bond should becapable of being disrupted under conditions suitable for carrying outthe claimed method. The dissociation rate constant (k_(off)) for thebinding between said first binding partner C1 and said first bindingsites Z1 can, for example, be as defined herein for a reversiblebinding, e.g. in the range of about 5×10⁻⁴ sec⁻¹. Also the k_(off) ofthis bond can be determined by any suitable means, for example, byfluorescence titration, equilibrium dialysis or surface plasmonresonance. The dissociation/removal of the staining of themultimerization reagent from the cells results in the removal of thedissociated receptor binding reagent, and thus of the whole multivalentbinding complex including the detectable label from the previouslystained cell.

According to the invention, the receptor binding reagent may be selectedsuch that it comprises at least one first binding partner C1 and thefirst multimerization reagent comprises at least two first binding sitesZ1, at least three or at least four first binding sites Z1 for the firstbinding partner C1. Alternatively, it is possible to use two different(kinds of) receptor binding reagents.

According to the invention, the partners can be chosen from thefollowing group: (a) said first binding partner C1 comprises astreptavidin or avidin binding peptide and said first multimerizationreagent comprises streptavidin, or avidin, or a streptavidin analog, oran avidin analog that reversibly binds to said streptavidin or avidinbinding peptide; or (b) said first binding partner C1 comprises a biotinanalog that reversibly binds to streptavidin or avidin and said firstmultimerisation reagent comprises streptavidin, or avidin, or astreptavidin analog, or an avidin analog that reversibly binds to saidbiotin analog. Said first binding partner C1 may comprise thestreptavidin-binding peptide Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:01) and said multimerization reagent comprises the streptavidin analoghaving the amino acid sequence Val⁴⁴-Thr⁴⁵-Ala⁴⁶-Arg⁴⁷ (SEQ ID NO: 02)at positions 44 to 47 of the wild-type streptavidin sequence or thestreptavidin analog having the amino acid sequenceIle⁴⁴-Gly⁴⁵-Ala⁴⁶-Arg⁴⁷ (SEQ ID NO: 03) at positions 44 to 47 of thewild-type streptavidin sequence. Both these muteins are described inU.S. Pat. No. 6,103,493, for example, and are commercially availableunder the trademark Strep-Tactin® from IBA GmbH, Gottingen, Germany. Thestreptavidin binding peptides might, for example, be single peptidessuch as the “Strep-tag®” described in U.S. Pat. No. 5,506,121, forexample, or streptavidin binding peptides having a sequentialarrangement of two or more individual binding modules as described inInternational Patent Publication WO 02/077018. Examples of streptavidinbinding peptides having a sequential arrangement of two or moreindividual binding modules include the di-tag3 sequence(WSHPQFEKGGGSGGGSGGGSWSHPQFEK; SEQ ID NO: 11), the di-tag2 sequenceTrp-Ser-His-Pro-Gln-Phe-Glu-Lys-(GlyGlyGlySer)₂-Trp-Ser-His-Pro-Gln-Phe-Glu-Lys(SEQ ID NO: 12) that are described in International Patent ApplicationWO02/077018 or U.S. Pat. No. 7,981,632 or the sequenceWSHPQFEKGGGSGGGSGGSAWSHPQFEK (SEQ ID NO: 13, also known asTwin-Strep-tag®).

The present invention also encompasses that the binding between thefirst binding partner C1 and said at least 2 first binding sites Z1 ofsaid multimerization reagent may occur in the presence of a divalentcation. In an illustrative example, the first binding partner C1comprises a calmodulin binding peptide and the multimerization reagentcomprises multimeric calmodulin as described in U.S. Pat. No. 5,985,658,for example. Alternatively, the first binding partner C1 may comprise aFLAG peptide and said first multimerization reagent may comprise anantibody binding to the FLAG peptide, e.g. the FLAG peptide, which bindsto the monoclonal antibody 4E11 as described in U.S. Pat. No. 4,851,341.In yet another illustrative example, the first binding partner C1comprises an oligohistidine tag and the first multimerization reagentcomprises an antibody or a transition metal ion binding theoligohistidine tag. The disruption of all these binding complexes may beaccomplished by metal ion chelation, e.g. calcium chelation, e.g. byaddition of EDTA or EGTA. Calmodulin, antibodies such as 4E11 orchelated metal ions or free chelators may be multimerized byconventional methods, e.g. by biotinylation and complexation withstreptavidin or avidin or multimers thereof or by the introduction ofcarboxyl residues into a polysaccharide, e.g. dextran, essentially asdescribed in Noguchi, A., Takahashi, T., Yamaguchi, T., Kitamura, K.,Takakura, Y., Hashida, M. & Sezaki, H. (1992). “Preparation andproperties of the immunoconjugate composed of anti-human colon cancermonoclonal antibody and mitomycin C dextran conjugate. BioconjugateChemistry 3, 132-137” in a first step and coupling of calmodulin orantibodies or chelated metal ions or free chelators via primary aminogroups to the carboxyl groups in the polysaccharide, e.g. dextran,backbone using conventional carbodiimide chemistry in a second step.

In the method of the invention, it is also possible that themultimerization reagent is an oligomer or a polymer of streptavidin oravidin or of any analog of streptavidin or avidin. The oligomer orpolymer may be crosslinked by a polysaccharide. Oligomers or polymers ofstreptavidin or of avidin or of analogs of streptavidin or of avidin maybe prepared by the introduction of carboxyl residues into apolysaccharide, e.g. dextran, essentially as described in “Noguchi, A.,Takahashi, T., Yamaguchi, T., Kitamura, K., Takakura, Y., Hashida, M. &Sezaki, H. (1992). Preparation and properties of the immunoconjugatecomposed of anti-human colon cancer monoclonal antibody and mitomycin Cdextran conjugate. Bioconjugate Chemistry 3,132-137” in a first step.Then streptavidin or avidin or analogs thereof may be coupled viaprimary amino groups of internal lysine residue and/or the freeN-terminus to the carboxyl groups in the dextran backbone usingconventional carbodiimide chemistry in a second step. However,cross-linked oligomers or polymers of streptavidin or avidin or of anyanalog of streptavidin or avidin may also be obtained by crosslinkingvia bifunctional linkers such as glutardialdehyde or by other methodsdescribed in the literature.

The invention further encompasses that the first binding partner C1 maycomprise an antigen and the multimerization reagent may comprise anantibody or antibody fragment against said antigen. The antigen may, forexample, be an epitope tag. Examples of suitable epitope tags include,but are not limited to, FLAG-tag (sequence: DYKDDDDK, SEQ ID NO: 04),Myc-tag (sequence: EQKLISEEDL, SEQ ID NO: 05), HA-tag (sequence:YPYDVPDYA, SEQ ID NO: 06), VSV-G-tag (sequence: YTDIEMNRLGK, SEQ ID NO:07), HSV-tag (sequence: QPELAPEDPED, SEQ ID NO: 08), and V5-tag(sequence: GKPIPNPLLGLDST, SEQ ID NO: 09). The antigen may also be aprotein, for example, the first binding partner C1 may comprise maltosebinding protein (MBP), chitin binding protein (CBP) or thioredoxin as anantigen. In these cases, the complex formed between the at least twofirst binding sites Z1 of the multimerization reagent (antibody) and theantigen can be disrupted by adding the free antigen, i.e. the freepeptide such as a Myc-tag or the HA-tag (epitope tag) or the freeprotein (such as MBP or CBP). In this context, it is noted that in casethe FLAG-tag is used as first binding partner C1 and the firstmultimerization reagent comprises an antibody or antibody fragmentbinding the FLAG tag, it is also possible of disrupting this reversiblebond by addition of the free FLAG peptide.

The invention also encompasses that the first binding partner C1 maycomprise glutathione S-transferase (GST) and said first multimerizationreagent may comprise glutathione as first binding site Z1 or whereinsaid first binding partner C1 may comprise a glutathione and said firstmultimerization reagent may comprises glutathione S-transferase as firstbinding site Z1. Here, the bond between the GST and glutathione can bedissociated by addition of excess glutathione. The free glutathione maycompetitively displace the glutathione comprised in C1 or Z1 that isbound to GST, allowing the first receptor binding agent to dissociatefrom the first multimerization reagent.

The invention also encompasses that the first binding partner C1 maycomprise an immunoglobulin Fc portion and said first multimerizationreagent may comprise a protein selected from the group consisting ofprotein A, protein G, protein a/g, and protein L, or wherein said firstbinding partner C1 as first binding site Z1 comprises a protein selectedfrom the group consisting of protein A, protein G, protein a/g, andprotein L and said first multimerization reagent comprises animmunoglobulin Fc portion as first binding site Z1. The bond between theimmunoglobulin Fc portion and the protein A, protein G, protein a/g, orprotein L may e.g. be disrupted by applying an acidic pH.

The invention further contemplates that the second receptor binding siteB2 comprised in the second receptor binding reagent or comprised in themultimeric receptor binding reagent M2 can generally be any compoundthat is defined herein for the first receptor binding reagent B1. It ispreferred that B2 is a MHC molecule. It is further encompassed that thesecond receptor binding site B2 may preferably be the same as the firstreceptor binding site B1. The second receptor binding site B2 may alsobe an antibody, a divalent antibody fragment, a monovalent antibodyfragment, and a proteinaceous binding molecule with antibody-likebinding.

Examples for a divalent antibody fragment comprise, but are not limitedto divalent antibody fragment is an (Fab)₂′-fragment, or a divalentsingle-chain Fv fragment. Examples of monovalent antibody fragmentsinclude, but are not limited to an Fab fragment, an Fv fragment, asingle domain antibody, and a single-chain Fv fragment (scFv).

Examples of proteinaceous binding molecules with antibody-like bindingproperties that can be used as receptor binding reagent thatspecifically binds the receptor molecule include, but are not limitedto, an aptamer, a mutein based on a polypeptide of the lipocalin family,a glubody, a protein based on the ankyrin scaffold, a protein based onthe crystalline scaffold, an adnectin, an avimer, a EGF-like domain, aKringle-domain, a fibronectin type I domain, a fibronectin type IIdomain, a fibronectin type III domain, a PAN domain, a Gla domain, aSRCR domain, a Kunitz/Bovine pancreatic trypsin Inhibitor domain,tendamistat, a Kazal-type serine protease inhibitor domain, a Trefoil(P-type) domain, a von Willebrand factor type C domain, anAnaphylatoxin-like domain, a CUB domain, a thyroglobulin type I repeat,LDL-receptor class A domain, a Sushi domain, a Link domain, aThrombospondin type I domain, an immunoglobulin domain or a animmunoglobulin-like domain (for example, domain antibodies or camelheavy chain antibodies), a C-type lectin domain, a MAM domain, a vonWillebrand factor type A domain, a Somatomedin B domain, a WAP-type fourdisulfide core domain, a F5/8 type C domain, a Hemopexin domain, an SH2domain, an SH3 domain, a Laminin-type EGF-like domain, a C2 domain,“Kappabodies” (Ill. et al. “Design and construction of a hybridimmunoglobulin domain with properties of both heavy and light chainvariable regions” Protein Eng 10:949-57 (1997)), “Minibodies” (Martin etal. “The affinity-selection of a minibody polypeptide inhibitor of humaninterleukin-6” EMBO J 13:5303-9 (1994)), “Janusins” (Traunecker et al.“Bispecific single chain molecules (Janusins) target cytotoxiclymphocytes on HIV infected cells” EMBO J 10:3655-3659 (1991) andTraunecker et al. “Janusin: new molecular design for bispecificreagents” Int J Cancer Suppl 7:51-52 (1992), a nanobody, a adnectin, atetranectin, a microbody, an affilin, an affibody or an ankyrin, acrystallin, a knottin, ubiquitin, a zinc-finger protein, anautofluorescent protein, an ankyrin or ankyrin repeat protein or aleucine-rich repeat protein, an avimer (Silverman, Lu Q, Bakker A, To W,Duguay A, Alba B M, Smith R, Rivas A, Li P, Le H, Whitehorn E, Moore KW, Swimmer C, Perlroth V, Vogt M, Kolkman J, Stemmer W P 2005, NatBiotech, December; 23(12):1556-61, E-Publication in Nat Biotech. 2005Nov. 20 edition); as well as multivalent avimer proteins evolved by exonshuffling of a family of human receptor domains as also described inSilverman J, Lu Q, Bakker A, To W, Duguay A, Alba B M, Smith R, Rivas A,Li P, Le H, Whitehorn E, Moore K W, Swimmer C, Perlroth V, Vogt M,Kolkman J, Stemmer W P, Nat Biotech, December; 23(12):1556-61,E-Publication in Nat. Biotechnology. 2005 Nov. 20 edition).

The invention further encompasses that the irreversible receptor bindingsite B3 comprised in the irreversible receptor binding reagent I2 may bean antibody, an divalent antibody fragment, a monovalent antibodyfragment, and a proteinaceous binding molecule with antibody-likebinding. It is understood that the irreversible receptor binding site B3is preferably not the same compound as the first receptor binding siteB1.

The invention further encompasses that the second binding partner C2 maycomprise biotin or a biotin analog and said second multimerizationreagent may comprise a streptavidin analog or an avidin analog as secondbinding site Z2 that essentially irreversibly binds to biotin or saidbiotin analog. As an illustrative example, the second binding partner C2may be biotin and the second binding site Z2 may be streptavidin. At thesame time, the first binding partner B1 may comprise astreptavidin-binding peptide as set forth in SEQ ID NO: 01 while thefirst binding site Z1 of the first multimerization reagent may comprisethe streptavidin analog having the amino acid sequenceVal⁴⁴-Thr⁴⁵-Ala⁴⁶-Arg⁴⁷ (SEQ ID NO: 02) at positions 44 to 47 of thestreptavidin wild type sequence or the streptavidin analog having theamino acid sequence Ile⁴⁴-Gly⁴⁵-Ala⁴⁶-Arg⁴⁷ (SEQ ID NO: 03) at thesesequence positions 44 to 47. Here, when contacting the multimericcomplexes with excess free biotin, the streptavidin-binding peptidecomprised in C1 is displaced from the first binding site Z1, while thebiotin comprised in C2 remains bound to the second binding site Z2. Thefirst multimerization reagent may thus dissociate from the target cellthus allowing measurement of the k_(off) rate of receptor and the firstreceptor binding site B1, while the second multimerization reagent staysattached to the target cell.

Alternatively, the second binding partner C2 and the second binding siteZ2 may be any pair of compounds that are defined for C1 and Z1, with theproviso that the bond between C2 and Z2 cannot disrupted by the samemethod, by which the bond between C1 and Z1 can be disrupted. As anillustrative example, C1 may be a strep-tag and Z1 may be streptactin,while C2 may be an oligohistidine tag and Z2 is a compound thatcomprises a divalent cation. If biotin is added to these complexes, thebond between C1 and Z1 will be disrupted, while the bond between C2 andZ2 will stay intact. Hence, although the bond C2 and Z2 can generally bedisrupted (e.g. by contacting the complex with a metal-chelating agent,such as EDTA), it can still serve as an irreversible multimer in themethods of the invention, as long as the bond between C2 and Z2 is notdisrupted by the same means or methods, by which the bond between C1 andZ1 can be disrupted.

The methods of the invention may comprise a step of contacting thetarget cell that comprises a receptor molecule R with a first receptorbinding reagent and said first multimerization reagent. Contacting thesethree components with each other can be conducted in any order. As anillustrative example, the first receptor binding agent may first becontacted with the first multimerization reagent to allow the formationof complexes of first receptor binding agent and first multimerizationreagent and then contacting the two compounds with the target cell. Asanother illustrative example, the cell can be contacted first with thefirst receptor binding reagent, while the first multimerization reagentis subsequently be contacted with the cell and the first receptorbinding reagent. As a further illustrative example, all three componentsmay be contacted with each other simultaneously.

The method of the present invention may further comprise the step ofcontacting the target cell with a second receptor binding agent and asecond multimerization reagent. Again, contacting these three componentscan be conducted in any order and the illustrative examples given forcontacting the target cell with the first receptor binding reagent andthe first multimerization reagent apply mutatis mutandis. Alternatively,the method of the present invention may further comprise the step ofcontacting the target cell with a multimeric receptor binding agent asdescribed herein, or contacting the target cell with an irreversiblereceptor binding reagent as described herein. The step of contacting thetarget cell with the first receptor binding reagent and the firstmultimerization reagent may preferably be performed prior to contactingthe cell with the second receptor binding agent and the secondmultimerization reagent or the multimeric receptor binding reagent orthe irreversible receptor binding reagent. Optionally, a washing stepcan be conducted between these two steps.

The methods of the present invention may further comprise a step ofdisrupting the binding between the first receptor binding reagent andthe first multimerization reagent. It is understood that this step maybe carried out after the cell has been contacted with the first receptorbinding reagent and the first multimerization reagent as well as thesecond receptor binding agent and the second multimerization reagent orthe multimeric receptor binding reagent or the irreversible receptorbinding reagent. Disruption of the binding between the first receptorbinding reagent and the first multimerization reagent can be conductedby any suitable method known to the skilled person or described herein.For example, the binding can be disrupted by contacting the boundcomplex with a competition reagent CR. Here, the competition reagent CRmay be capable to compete with first binding partner C1 for the bindingto the first binding site Z1 on the first multimerization reagent.Suitable competition reagents are described herein and depend on thetype of what first binding partner C1 is comprised in the first receptorbinding reagent and what first binding site Z1 is comprised in the firstmultimerization reagent. As an illustrative example, where the firstbinding partner C1 is a streptavidin binding peptide and the firstmultimerization reagent may be a streptavidin mutein such asStrep-tactin®, the competition reagent CR may biotin or a biotin analog.

The binding between the first receptor binding reagent and the firstmultimerization reagent may also be conducted by metal ion chelation,e.g. by contacting the complex with a metal-chelating agent, such asEDTA or EGTA. This type of disrupting the binding reagent and the firstmultimerization reagent may also be conducted by metal ion chelation mayfor example be conducted if the first binding partner C1 is aoligohistidine tag and the first binding site Z1 comprises a divalentcation. Another method of disrupting the binding between the firstreceptor binding reagent and the first multimerization reagent is by pHshift. This method may for example be conducted if the first bindingsite Z1 comprises an immunoglobulin Fc region and the first binding siteZ1 comprises a protein A.

In a preferred embodiment, the target cell is a T cell, preferably aCD8+ T cell; the receptor R is a T cell receptor; the first receptorbinding site B1 and the second receptor binding site B2 are the same andare both a MHC molecule; the first binding partner C1 comprises astreptavidin binding peptide and the first multimerization reagentcomprises streptactin; the second binding partner comprises a biotin andthe second multimerization reagent comprises streptavidin, a firstdetectable label (e.g. Alexa 488) is bound to the first receptor bindingreagent, a second detectable (e.g. BV421) label is preferably bound tothe second multimerization reagent; a third detectable label (e.g. APC)is preferably attached to the first multimerization reagent. It isunderstood that first, second, and the optional third detectable labelcan be distinguished from each other. Here, biotin can be used as acompetition reagent CR for disrupting the reversible binding between C1and Z1 of the steptactin.

The methods of the present invention may further comprise the step ofdetecting the first detectable label attached to the target cell anddetecting the second detectable label attached to the target cell. Here,detecting both detectable labels may preferably be conducted after thestep of disrupting the binding between the first receptor bindingreagent and the first multimerization reagent. In cases where the firstmultimerization reagent comprises a third detectable label, the methodsof the present invention may further comprise detecting the thirddetectable label. In addition, the methods of the present invention maycomprise detecting (a) further detectable label(s). As an illustrativeexample, the target cell may be additionally stained with a CD8 antibodywith a further detectable label, such as e.g. eF450. The target cell mayalso be stained with a dye that allows for discrimination between viableand dead cell. An illustrative example for such a dye is propidiumiodide, which is an intercalating agent and a fluorescent molecule thatis membrane impermeant and generally excluded from viable cells andwhich can thus be used for identifying dead cells.

It is contemplated by the invention that the detection of the detectablelabels may be conducted by a flow cytometry based analysis. Flowcytometry based analysis is typically combined with optical detection toidentify and classify cells and allows speed combined with highsensitivity and specificity. It allows a simultaneous multiparametricanalysis of the physical and chemical characteristics of single cellsflowing through an optical or electronic detection device. Thesespecific physical and chemical characteristics may comprise the specificlight scattering and/or fluorescent characteristics of each cell.

The present invention encompasses the use of two detectable labels thatare directly or indirectly bound to a receptor molecule R. While thefirst detectable label is reversibly bound to the cell, binding of thesecond detectable label to the target cell/receptor molecule R isessentially irreversible. Thus, detection of a signal of the seconddetectable label may be indicative for the presence of the receptormolecule R on the target cell. While the presence of the seconddetectable label may be indicative for the presence of the receptormolecule R on the target cell, the presence or absence of the firstdetectable label on said cell may be indicative for the non-dissociationor the dissociation of the first receptor binding site B1 from thereceptor molecule. Here, presence of the first detectable label isindicative for the non-dissociation of the first receptor binding sitewhile the absence is indicative for the dissociation of the same. It isunderstood that the reduction of detection events of the firstdetectable label on a cell on which the second detectable signal isdetected may be indicative for the kinetic of dissociation of the firstreceptor binding site B1 from the receptor molecule R. Such dissociationmay follow the kinetic of an exponential decay. In analyzing the numberof detection events for the first detectable label on a cell, where thesecond detectable label is present, the dissociation rate constant(k_(off)) for the binding of the receptor molecule R and the firstreceptor binding site B1 may be obtained by standard methods that arefamiliar to the skilled person (e.g. curve fitting).

The methods of the invention may generally allow for the determinationof any k_(off) values of the binding of a receptor molecule R on atarget cell and a first receptor binding site B1. However, the method ispreferably applied for a binding of a receptor molecule R on a targetcell and a first receptor binding site B1, where the k_(off) value issuspected to be within a range of about 10⁰ sec⁻¹ to about 10⁻⁴ sec⁻¹,preferably within a range of 10⁻¹ sec⁻¹ to about 10⁻³ sec⁻¹

The methods of the invention can be carried out at any suitabletemperature. Typically, the contacting of the mixture containing thetarget cell with any of the first receptor binding reagent, the firstmultimerization reagent, the second receptor binding reagent, the secondmultimerization reagent, the multimeric receptor binding reagent M2 orthe irreversible receptor binding reagent I2 and later the disruption ofthe binding between the first receptor binding reagent and the firstmultimerization reagent and also the detection of the any one of thefirst detectable label, the second detectable label or the thirddetectable label may be carried out at such temperatures, at whichsubstantially no activation and/or no signaling events occur, whichmight result in an alteration of the target cell, e.g. the T cellphenotype, in case a T cell is to be stained or isolated. The methods ofthe present invention or each individual step of the methods of theinvention may thus be preferably carried out at a temperature of ≤15° C.or carried out at a temperature of ≤4° C.

The invention further encompasses that the target cell may be comprisedin a sample. The sample may comprise the target cell and a plurality ofother cells. The sample may comprise a population of cells (e.g. CD8+ Tcells) and the target cell may be a subpopulation thereof (e.g. CD8+ Tcells specific for a certain antigen).

The sample may be from any suitable source, typically all sample of abody tissue or a body fluid such as blood. The sample may thus beperipheral blood sample. In the latter case, the sample might forexample, be a population of peripheral blood mononuclear cells (PBMC)that can be obtained by standard isolation methods such as Ficollgradient of blood cells. The cell population comprised in the sample mayhowever also be in purified form and might have been isolated using areversible cell staining/isolation technology as described patent inU.S. Pat. Nos. 7,776,562, 8,298,782, International Patent applicationWO02/054065 or International Patent Application WO2013/011011.Alternatively, the population of cells can also be obtained by cellsorting via negative magnetic immunoadherence as described in U.S. Pat.No. 6,352,694 B1 or European Patent EP 0 700 430 B1. If an isolationmethod described here is used in basic research, the sample might becells of in vitro cell culture experiments. The sample will typicallyhave been prepared in form of a fluid, such as a solution or dispersion.

The sample may be obtained from a subject. A “subject” as used herein,refers to a human or non-human animal, generally a mammal. A subject maybe a mammalian species such as a rabbit, a mouse, a rat, a guinea pig, ahamster, a dog, a cat, a pig, a cow, a goat, a sheep, a horse, a monkey,an ape or, preferably, a human. While a subject is typically a livingorganism, the sample may also be taken post-mortem.

The present invention also encompasses a cell comprising at least threereceptor molecules R, wherein the cell has bound to at least tworeceptor molecules R (i) a first receptor binding reagent, the firstreceptor binding reagent comprising at least one first receptor bindingsite B1, wherein the first receptor binding site B1 specifically bindsto said receptor molecule R, the first receptor binding reagent furthercomprising at least one first binding partner C1, wherein the firstbinding partner C1 is capable of being reversibly bound to a firstbinding site Z1 of a first multimerization reagent, and a firstmultimerization reagent, the first multimerization reagent comprising atleast two first binding sites Z1 for the reversible binding of the firstbinding partner C1 of the first receptor binding reagent. The firstreceptor binding reagent (i) and the first multimerization reagent (ii)form a first multivalent binding complex that binds to said cell, thefirst multivalent binding complex comprising at least two of said firstreceptor binding reagents bound to one said first multimerizationreagent. It is understood that the first detectable label is bound orcapable of binding to said first receptor binding reagent. The cell thushas a first detectable label attached to it. Further, the cell has asecond detectable label attached to it. While the first detectable labelmay be reversibly bound to the cell, the second detectable label may beessentially irreversibly attached to the cell. It is also understoodthat the first detectable label and the second detectable label aredifferent from each other, meaning that they are not the same compound.The first multimerization reagent optionally further comprises a thirddetectable label. It is understood that each of the first, second, andthird detectable label are preferably different from each other and canpreferably be distinguished from each other. Such a cell is especiallyuseful for determining the dissociation rate constant (k_(off)) of thereceptor molecule R and the first receptor binding site B, which can beperformed according to the methods of the invention.

As discussed, the cell has a second detectable label attached to it,wherein the second detectable label is essentially irreversibly attachedto the receptor molecule R. It is understood that there are multiplepossibilities for essentially irreversibly attaching such said secondlabel to a one or more receptor molecule(s) R. The invention thusencompasses, as one possibility, that the second detectable label isattached to the cell via an irreversible multimer according to theinvention. Hence, the cell may have further bound to at least tworeceptor molecules R (iii) a second receptor binding reagent, the secondreceptor binding reagent comprising at least one second receptor bindingsite B2, wherein the second receptor binding site B2 specifically bindsto said receptor molecule R, the second receptor binding reagent furthercomprising at least one second binding partner C2, wherein the secondbinding partner C2 is capable of being stably bound to a second bindingsite Z2 of a second multimerization reagent, and (iv) a secondmultimerization reagent, the second multimerization reagent comprisingat least two second binding sites Z2 for the stable binding of thesecond binding partner C2 of the second receptor binding reagent,wherein the second receptor binding reagent (iii) and the secondmultimerization reagent (iv) form a second multivalent binding complexthat binds to said target cell, the second multivalent binding complexcomprising at least two of second receptor binding reagents bound to onesaid second multimerization reagent; and wherein said second detectablelabel is bound to said second multivalent binding complex.

Alternatively, the cell may have attached to it a multimeric receptorbinding reagent M2 according to the invention. Hence, the cell may havefurther bound to at least two receptor molecules R (iii′) a multimericreceptor binding reagent M2, the multimeric second receptor bindingreagent comprising at least two second receptor binding sites B2,wherein the second receptor binding site B2 specifically binds to saidreceptor molecule R, wherein the multimeric receptor binding reagent M2essentially irreversibly binds to said cell via said at least tworeceptor molecules R, wherein said second detectable label is bound tosaid multimeric receptor binding reagent M2.

As another alternative, the cell may have attached to it an irreversiblereceptor binding reagent I2 according to the invention. Hence, the cellmay have further bound to a receptor molecule R (iii″) an irreversiblereceptor binding reagent I2, wherein the irreversible receptor bindingreagent I2 specifically binds to said receptor molecule R, wherein theirreversible receptor binding reagent I2 essentially irreversibly bindsto said receptor molecule R, wherein said second detectable label isbound to said irreversible receptor binding reagent I2

It is understood that the cell may be any target cell according to theinvention. Likewise, the receptor molecule R may be any receptormolecule R according to the invention.

The invention further encompasses a kit suitable for conducting themethods of the invention. The kit may thus comprise (i) a first receptorbinding reagent, the first receptor binding reagent comprising at leastone first receptor binding site B1, wherein the first receptor bindingsite B1 specifically binds to said receptor molecule R, the firstreceptor binding reagent further comprising at least one first bindingpartner C1, wherein the first binding partner C1 is capable of beingreversibly bound to a first binding site Z1 of a first multimerizationreagent, and (ii) a first multimerization reagent, the firstmultimerization reagent comprising at least two first binding sites Z1for the reversible binding of the first binding partner C1 of the firstreceptor binding reagent, wherein the first receptor binding reagent (i)and the first multimerization reagent (ii) are capable of forming afirst multivalent binding complex that is capable of binding to saidtarget cell, wherein the first multivalent binding complex may compriseat least two of said first receptor binding reagents bound to one saidfirst multimerization reagent, wherein a first detectable label is boundor capable of binding to said first receptor binding reagent.

The kit may further comprise (iii) a second receptor binding reagent,the second receptor binding reagent comprising at least one secondreceptor binding site B2, wherein the second receptor binding site B2specifically binds to said receptor molecule R, the second receptorbinding reagent further comprising at least one second binding partnerC2, wherein the second binding partner C2 is capable of being stablybound to a second binding site Z2 of a second multimerization reagent,and (iv) a second multimerization reagent, the second multimerizationreagent comprising at least two second binding sites Z2 for the stablebinding of the second binding partner C2 of the second receptor bindingreagent, wherein the second receptor binding reagent (iii) and thesecond multimerization reagent (iv) are capable of forming a secondmultivalent binding complex that is capable of binding to said targetcell, the second multivalent binding complex comprising at least two ofsecond receptor binding reagents bound to one said secondmultimerization reagent; wherein a second detectable label is bound toor capable of binding to said second multivalent binding complex.

Alternatively, the kit may further comprise (iii′) a multimeric receptorbinding reagent M2, the multimeric second receptor binding reagentcomprising at least two second receptor binding sites B2, wherein thesecond receptor binding site B2 specifically binds to said receptormolecule R, wherein the multimeric receptor binding reagent M2 iscapable of essentially irreversibly binding to said target cell via saidreceptor molecule R, wherein a second detectable label is bound to saidmultimeric receptor binding reagent M2.

Alternatively, the kit may further comprise (iii″) an irreversiblereceptor binding reagent I2, wherein the irreversible receptor bindingreagent I2 specifically binds to said receptor molecule R, wherein theirreversible receptor binding reagent I2 is capable of essentiallyirreversibly binding to said receptor molecule R, wherein a seconddetectable label is bound to said irreversible receptor binding reagentI2.

The kit may further comprise a competition reagent CR, wherein thecompetition reagent CR is capable of competing with first bindingpartner C1 for the binding to the first binding site Z1 on the firstmultimerization reagent. The kit may further comprise a metal chelatingreagent, wherein the metal chelating reagent is preferably of EDTA orEGTA.

The invention further encompasses an apparatus. Such an apparatuscomprises a first container containing a target cell comprising areceptor molecule R, and (i) a first receptor binding reagent, the firstreceptor binding reagent comprising at least one first receptor bindingsite B1, wherein the first receptor binding site B1 specifically bindsto said receptor molecule R, the first receptor binding reagent furthercomprising at least one first binding partner C1, wherein the firstbinding partner C1 is capable of being reversibly bound to a firstbinding site Z1 of a first multimerization reagent, and a firstmultimerization reagent, the first multimerization reagent comprising atleast two first binding sites Z1 for the reversible binding of the firstbinding partner C1 of the first receptor binding reagent, wherein thefirst receptor binding reagent (i) and the first multimerization reagent(ii) are capable of forming a first multivalent binding complex thatbinds to said target cell, the first multivalent binding complexcomprising at least two of said first receptor binding reagents bound toone said first multimerization reagent; and wherein a first detectablelabel is bound or capable of binding to said first receptor bindingreagent; and wherein a second detectable label is essentiallyirreversibly attached to said receptor molecule R, wherein the firstdetectable label is not the second detectable label.

The apparatus further comprises a second container containing a fluidcomprising a competition reagent CR, wherein the competition reagent CRis capable of competing with first binding partner C1 for the binding tothe first binding site Z1 on the first multimerization reagent or ametal chelating reagent, wherein the metal chelating reagent ispreferably EDTA or EGTA. The first container and a second container areconnected such that a fluid can be transferred from the second containerto the first container.

The first container of the apparatus may further contain (iii) a secondreceptor binding reagent, the second receptor binding reagent comprisingat least one second receptor binding site B2, wherein the secondreceptor binding site B2 specifically binds to said receptor molecule R,the second receptor binding reagent further comprising at least onesecond binding partner C2, wherein the second binding partner C2 iscapable of being stably bound to a second binding site Z2 of a secondmultimerization reagent, and (iv) a second multimerization reagent, thesecond multimerization reagent comprising at least two second bindingsites Z2 for the stable binding of the second binding partner C2 of thesecond receptor binding reagent, wherein the second receptor bindingreagent (iii) and the second multimerization reagent (iv) are capable offorming a second multivalent binding complex that binds to said targetcell, the second multivalent binding complex comprising at least two ofsecond receptor binding reagents bound to one said secondmultimerization reagent; and wherein said second detectable label isbound to said second multivalent binding complex.

Alternatively, the first container of the apparatus may further contain(iii′) a multimeric receptor binding reagent M2, wherein the multimericsecond receptor binding reagent comprises at least two second receptorbinding sites B2, wherein the second receptor binding site B2specifically binds to said receptor molecule R, wherein the multimericreceptor binding reagent M2 is capable of essentially irreversiblybinding to said target cell via said receptor molecule R, wherein saidsecond detectable label is bound to said multimeric receptor bindingreagent M2.

Alternatively, the first container of the apparatus may further contain(iii″) an irreversible receptor binding reagent I2, wherein theirreversible receptor binding reagent I2 specifically binds to saidreceptor molecule R, wherein the irreversible receptor binding reagent12 is capable of essentially irreversibly binding to said receptormolecule R, wherein said second detectable label is bound to saidirreversible receptor binding reagent I2.

The apparatus may further comprise a device for controlling thetemperature of the first container. Said device may comprise anisolation layer that at least partially surrounds the first container.The apparatus may further comprise a thermometer which is capable ofdetermining the temperature within the first container. The device mayfurther comprise a cooling element. Such a cooling element may be an aircooler. The cooling element may allow for electronic control of thetemperature of or within the first container.

The first container may for example be a sample tube. Typical sampletubes may consist of plastic, such as polyethylene, polycarbonate,polystyrol or polypropylene, glass, or metal, such as steel. The tubemay have a cylindrical shape and a round, flat or conical base. Thevolume of the first container may typically have a volume in the rangeof microliters to liters, typically about 0.01 mL to about 1000 mL,preferably about 0.05 mL to about 500 mL, about 0.1 mL to about 200 mL,about 0.5 mL to about 100 mL, about 1 mL to about 50 mL, about 1 mL toabout 25 mL, including about 1, about 2, about 3, about 4, about 5,about 6, about 7, about 8, about 9, about 10, about 11, about 12, about13, about 14, about 15, about 16, about 17, about 18, about 19, about20, about 21, about 22, about 23, about 24, about 25 mL, including anynumber in between.

The second container may be a syringe. Any syringe can be comprisedwithin the apparatus of the invention. It can be made of any materialthat is typical for a syringe, including plastic, such as polyethylene,polycarbonate, polystyrol or polypropylene, glass, or metal, such assteel, or mixtures thereof. The syringe may be suitable for manualoperation or may be suitable for electronically controlled operation.

The apparatus of the invention may be comprised in an apparatus for flowcytometry. The first container of the apparatus may be connected with asample inlet of an apparatus for flow cytometry.

The first container and the second container may be connected via acannula or a tubing. A cannula may typically be a syringe needle and mayfor example be made of metal, such as copper, or (stainless) steel. Atubing may be of any material that is typically used for a tubing. Itmay e.g. consist of silicon, gum, pelytetrafluoroethylene (PTFE),polyvinyl chloride (PVC), copper, or (stainless) steel, just to name afew. The cannula or tubing may have an inner diameter of about 0.1 toabout 20 mm, typically about 0.2 to about 10 mm, about 0.5 to about 5mm, including about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm,about 0.9 mm, about 1 mm, about 1.2 about 1.5 mm, about 2 mm, about 3mm, about 4 mm, or about 5 mm.

A valve may be arranged within the connection between said firstcontainer and said second container. Such a valve may preferably a threeway valve. The three way valve may be connected to a third container.The third container may contain a reagent that is capable of disruptingthe binding between the first binding partner C1 and the first bindingsite Z1 comprised in the compounds that are contained in the firstcontainer. By using a three-way valve, the second container/syringe maybe loaded and unloaded without detaching or disconnecting the secondcontainer/syringe from the first container.

The invention also encompasses a method of isolating a high-avidity Tcell. This method may comprise a first step, in which the dissociationrate constant (k_(off)) of a T cell in a sample obtained from a subjectis determined according to the methods of the invention. Hereby, ahigh-avidity T cell may be identified. A “high-avidity T cell” may bedefined by the k_(off) value of the T cell receptor when binding to agiven antigen. The T cell may be of “high avidity”, if the k_(off) valueis equal or below a given threshold value. The threshold value maydepend on the purpose, for which the high-avidity T cell will beisolated for. Typically, the threshold value is in the range of about10⁻¹ sec⁻¹ to about 10⁻³ sec⁻¹ preferably, the threshold value may be inthe range of about 5×10⁻² sec⁻¹ to about 2×10⁻³ sec⁻¹, preferably about2×10⁻² sec⁻¹ to about 5×10⁻³ sec⁻¹, preferably about 1×10⁻² sec⁻¹.

The method may then comprise a further step of isolating said T cell orpopulation of T cells from a sample obtained from the same subject.Isolation of said T cell (population) can be performed by any methodknow in the art, for example by using a reversible cellstaining/isolation technology as described patent in U.S. Pat. Nos.7,776,562, 8,298,782, International Patent application WO02/054065 orInternational Patent Application WO2013/011011. The sample, out of whichthe T cell (population) is isolated in the second step may be the samesample as in the first step or may be another sample obtained from thesame subject.

The present invention is further characterized by the following items

Item 1. A method of determining the dissociation rate constant (k_(off))of a receptor molecule R on a target cell and a first receptor bindingsite B1, comprising detecting a first detectable label attached to thetarget cell and a second detectable label attached to the target cell,wherein the cell has been contacted with (i) a first receptor bindingreagent, the first receptor binding reagent comprising at least onefirst receptor binding site B1, wherein the first receptor binding siteB1 specifically binds to said receptor molecule R, the first receptorbinding reagent further comprising at least one first binding partnerC1, wherein the first binding partner C1 is capable of being reversiblybound to a first binding site Z1 of a first multimerization reagent, and(ii) a first multimerization reagent, the first multimerization reagentcomprising at least two first binding sites Z1 for the reversiblebinding of the first binding partner C1 of the first receptor bindingreagent, wherein the first receptor binding reagent (i) and the firstmultimerization reagent (ii) form a first multivalent binding complexthat binds to said target cell, the first multivalent binding complexcomprising at least two of said first receptor binding reagents bound toone said first multimerization reagent; and wherein said firstdetectable label is bound or capable of binding to said first receptorbinding reagent; and wherein said second detectable label is essentiallyirreversibly attached to said receptor molecule R, wherein the firstdetectable label and the second detectable label are different from eachother.

Item 2. The method of item 1, wherein the cell has further beencontacted with (iii) a second receptor binding reagent, the secondreceptor binding reagent comprising at least one second receptor bindingsite B2, wherein the second receptor binding site B2 specifically bindsto said receptor molecule R, the second receptor binding reagent furthercomprising at least one second binding partner C2, wherein the secondbinding partner C2 is capable of being stably bound to a second bindingsite Z2 of a second multimerization reagent, and (iv) a secondmultimerization reagent, the second multimerization reagent comprisingat least two second binding sites Z2 for the stable binding of thesecond binding partner C2 of the second receptor binding reagent,wherein the second receptor binding reagent (iii) and the secondmultimerization reagent (iv) form a second multivalent binding complexthat binds to said target cell, the second multivalent binding complexcomprising at least two of second receptor binding reagents bound to onesaid second multimerization reagent; and wherein said second detectablelabel is bound to said second multivalent binding complex.

Item 3. The method of item 1, wherein the cell has further beencontacted with (iii′) a multimeric receptor binding reagent M2, themultimeric second receptor binding reagent comprising at least twosecond receptor binding sites B2, wherein the second receptor bindingsite B2 specifically binds to said receptor molecule R, wherein themultimeric receptor binding reagent M2 essentially irreversibly binds tosaid target cell via said receptor molecule R, wherein said seconddetectable label is bound to said multimeric receptor binding reagentM2.

Item 4. The method of item 1, wherein the cell has further beencontacted with (iii″) an irreversible receptor binding reagent I2,wherein the irreversible receptor binding reagent I2 specifically bindsto said receptor molecule R, wherein the irreversible receptor bindingreagent I2 essentially irreversibly binds to said receptor molecule R,wherein said second detectable label is bound to said irreversiblereceptor binding reagent I2.

Item 5. The method of any one of the preceding items, wherein saidreceptor molecule R is a T cell receptor (TCR).

Item 6. The method of any one of the preceding items, wherein said firstreceptor binding site B1 is a MHC molecule.

Item 7. The method of any one of the preceding items, wherein (a) saidfirst binding partner C1 comprises a streptavidin or avidin bindingpeptide and said first multimerization reagent comprises streptavidin,or avidin, or a streptavidin analog, or an avidin analog that reversiblybinds to said streptavidin or avidin binding peptide; or (b) said firstbinding partner C1 comprises a biotin analog that reversibly binds tostreptavidin or avidin and said first multimerisation reagent comprisesstreptavidin, or avidin, or a streptavidin analog, or an avidin analogthat reversibly binds to said biotin analog.

Item 8. The method of item 7, wherein said first binding partner C1comprises the streptavidin-binding peptideTrp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 01) and said multimerizationreagent comprises the streptavidin analog Val⁴⁴-Thr⁴⁵-Ala⁴⁶-Arg⁴⁷ (SEQID NO: 02) or the streptavidin analog Ile⁴⁴-Gly⁴⁵-Ala⁴⁶-Arg⁴⁷ (SEQ IDNO: 03).

Item 9. The method of any one of items 1 to 6, wherein the bindingbetween said first binding partner C1 and said first binding site Z1 ofsaid first multimerization reagent occurs in the presence of a divalentcation.

Item 10. The method of item 9, wherein a. said first binding partner C1comprises a calmodulin binding peptide and the said firstmultimerization reagent comprises calmodulin, or b. said first bindingpartner C1 comprises an oligohistidine tag and said firstmultimerization reagent comprises a metal ion bound to a metal chelatingagent.

Item 11. The method of any one of items 1 to 6, wherein said firstbinding partner C1 comprises an antigen and said first multimerizationreagent comprises an antibody against said antigen.

Item 12. The method of item 11, wherein the antigen is an epitope tag.

Item 13. The method of item 18, where the epitope tag is selected fromthe group consisting of FLAG-tag (sequence: DYKDDDDK, SEQ ID NO: 04),Myc-tag (sequence: EQKLISEEDL, SEQ ID NO: 05), HA-tag (sequence:YPYDVPDYA, SEQ ID NO: 06), VSV-G-tag (sequence: YTDIEMNRLGK, SEQ ID NO:07), HSV-tag (sequence: QPELAPEDPED, SEQ ID NO: 08), and V5-tag(sequence: GKPIPNPLLGLDST, SEQ ID NO: 09).

Item 14. The method of item 11, wherein said antigen comprises aprotein.

Item 15. The method of item 14, wherein the protein is selected from thegroup of the maltose binding protein (MBP), chitin binding protein (CBP)and thioredoxin.

Item 16. The method of any one of items 1 to 6, wherein said firstbinding partner C1 comprises glutathione S-transferase and said firstmultimerization reagent comprises glutathione or wherein said firstbinding partner C1 comprises a glutathione and said firstmultimerization reagent comprises glutathione S-transferase.

Item 17. The method of any one of items 1 to 6, wherein said firstbinding partner C1 comprises an immunoglobulin Fc portion and said firstmultimerization reagent comprises a protein selected from the groupconsisting of protein A, protein G, protein a/g, and protein L, orwherein said first binding partner C1 comprises a protein selected fromthe group consisting of protein A, protein G, protein a/g, and protein Land said first multimerization reagent comprises an immunoglobulin Fcportion.

Item 18. The method of any one of items 2, 3 and 5 to 17, wherein thesecond receptor binding site B2 is a MHC molecule.

Item 19. The method of any one of items 2, 3 and 5 to 18, wherein thefirst receptor binding site B1 and the second receptor binding site B2are the same.

Item 20. The method of any one of items 2 to 17, wherein the secondreceptor binding site B2 or irreversible receptor binding reagent I2 isselected from the group consisting of an antibody, an divalent antibodyfragment, a monovalent antibody fragment, and a proteinaceous bindingmolecule with antibody-like binding.

Item 21. The method of item 20, wherein the divalent antibody fragmentis an (Fab)₂′-fragment, or a divalent single-chain Fv fragment.

Item 22. The method of item 20, wherein the monovalent antibody fragmentis selected from the group consisting of a Fab fragment, an Fv fragment,and a single-chain Fv fragment (scFv).

Item 23. The method of item 20, wherein the proteinaceous bindingmolecule with antibody-like binding properties is selected from thegroup of an aptamer, a mutein based on a polypeptide of the lipocalinfamily, a glubody, a protein based on the ankyrin scaffold, a proteinbased on the crystalline scaffold, an adnectin, and an avimer.

Item 24. The method of any one of items 2 and 5 to 23, wherein saidsecond binding partner C2 comprises biotin or a biotin analog and saidsecond multimerization reagent comprises a streptavidin analog or anavidin analog that essentially irreversibly binds to biotin or saidbiotin analog.

Item 25. The method of any one of the preceding items, wherein saidtarget cell is a mammalian cell.

Item 26. The method of item 24, wherein said mammalian cell is alymphocyte or a stem cell.

Item 27. The method of item 25, wherein the lymphocyte is a T cell, aT-helper cell, a B cell or a natural killer cell.

Item 28. The method of item 26, wherein the T-cell is selected from thegroup of a CMV-specific CD8+ T-lymphocyte, a cytotoxic T-cell a, memoryT-cell and a regulatory T-cell.

Item 29. The method of any one of the preceding items, wherein the firstdetectable label is a fluorescent dye.

Item 30. The method of any one of the preceding items, wherein thesecond detectable label is a fluorescent dye.

Item 31. The method of any one of the preceding items, wherein each ofthe first detectable label is a first fluorescent dye and the seconddetectable label is a second fluorescent dye, wherein the emissionsignal of the first fluorescent dye can preferably be distinguished fromthe emission signal of the second fluorescent dye.

Item 32. The method any one of items 2 to 31, comprising (a) contactingsaid target cell comprising said receptor molecule R with (i) said firstreceptor binding reagent and (ii) said first multimerization reagent and(b) contacting said target cell with (iii) said second receptor bindingreagent and (iv) said second multimerization reagent, or contacting saidtarget cell with (iii′) said multimeric receptor binding reagent; orcontacting said target cell with (iii″) said irreversible receptorbinding reagent.

Item 33. The method item 32, wherein (a) is performed prior to (b).

Item 34. The method of item 33, further comprising after (a) and priorto (b) a washing step.

Item 35. The method of any one of items 32 to 34, further comprising (c)disrupting the binding between (i) the first receptor binding reagentand (ii) the first multimerization reagent.

Item 36. The method of item 35, wherein the binding between (i) thefirst receptor binding reagent and (ii) the first multimerizationreagent is disrupted by contacting (i) and (ii) with a competitionreagent CR, wherein the competition reagent CR is capable of competingwith first binding partner C1 for the binding to the first binding siteZ1 on the first multimerization reagent.

Item 37. The method of item 36, wherein the first binding partner C1 isa streptavidin binding peptide and the competition reagent CR is biotinor a biotin analog.

Item 38. The method of item 36 or 37, wherein the first multimerizationreagent comprises a streptavidin or a streptavidin analog.

Item 39. The method of item 35, wherein the binding between (i) thefirst receptor binding reagent and (ii) the first multimerizationreagent is disrupted by metal ion chelation.

Item 40. The method of item 39, wherein the metal chelation isaccomplished by addition of EDTA or EGTA.

Item 41. The method of item 35, wherein the binding between (i) thefirst receptor binding reagent and (ii) the first multimerizationreagent is disrupted by pH shift.

Item 42. The method of any one of items 32 to 41, further comprising (d)detecting the first detectable label attached to the target cell anddetecting the second detectable label attached to the target cell.

Item 43. The method of item 42, wherein the first detectable label is afirst fluorescent dye and the second detectable label is a secondfluorescent dye, wherein the emission signal of the first fluorescentdye can preferably be distinguished from the emission signal of thesecond fluorescent dye.

Item 44. The method of item 43, wherein the detection of the firstdetectable label and the second detectable label is by flow cytometry.

Item 45. The method of item 44, wherein the detection of seconddetectable signal is indicative for the presence of the receptormolecule R on the target cell.

Item 46. The method of item 45, wherein the detection of the firstdetectable label on a cell on which the second detectable signal isdetected is indicative for the non-dissociation of the of the firstreceptor binding site B1 from the receptor molecule R at the time ofdetecting the first detectable label.

Item 47. The method of item 45, wherein the non-detection of the firstdetectable label on a cell on which the second detectable signal isdetected is indicative for the dissociation of the of the first receptorbinding site B1 from the receptor molecule R at the time of detectingthe first detectable label.

Item 48. The method of any one of items 45 to 47, wherein the reductionof detection events of the first detectable label on a cell on which thesecond detectable signal is detected is indicative for the kinetic ofdissociation of the first receptor binding site B1 from the receptormolecule R.

Item 49. The method of any one of the preceding items, wherein thedissociation rate constant (k_(off)) for the reversible binding betweensaid binding site Z1 and said partner C1 is in the range of 0.5×10⁻⁴sec⁻¹ or greater.

Item 50. The method of any one of items 2, and 5 to 48, wherein thedissociation rate constant (k_(off)) for the binding between saidbinding site Z2 and said partner C2 is in the range of 1×10⁻⁵ sec⁻¹ orless.

Item 51. The method of any one of items 3 and 5 to 48, wherein thedissociation rate constant (k_(off)) for the essentially irreversiblebinding between said multimeric receptor binding reagent M2 and saidtarget cell is in the range of 1×10⁻⁵ sec⁻¹ or less.

Item 52. The method of any one of items 4 to 48, wherein the wherein thedissociation rate constant (k_(off)) for the essentially irreversiblebinding between said irreversible receptor binding reagent I2 and saidreceptor molecule R is in the range of 1×10⁻⁵ sec⁻¹ or less.

Item 53. The method of any one of the preceding items, wherein thedissociation rate constant (k_(off)) of a receptor molecule R on atarget cell and a first receptor binding site B1 is suspected to be in arange of 10° sec⁻¹ to 10⁻⁴ sec⁻¹.

Item 54. The method of any one of the preceding items, whereincontacting the cell with the first receptor binding reagent is carriedout at a temperature of <15° C.

Item 55. The method of item 54, wherein said contacting is carried outat a temperature of ≤4° C.

Item 56. A cell comprising at least three receptor molecules R, whereinthe cell has bound to at least two receptor molecules R (i) a firstreceptor binding reagent, the first receptor binding reagent comprisingat least one first receptor binding site B1, wherein the first receptorbinding site B1 specifically binds to said receptor molecule R, thefirst receptor binding reagent further comprising at least one firstbinding partner C1, wherein the first binding partner C1 is capable ofbeing reversibly bound to a first binding site Z1 of a firstmultimerization reagent, and (ii) a first multimerization reagent, thefirst multimerization reagent comprising at least two first bindingsites Z1 for the reversible binding of the first binding partner C1 ofthe first receptor binding reagent, wherein the first receptor bindingreagent (i) and the first multimerization reagent (ii) form a firstmultivalent binding complex that binds to said target cell, the firstmultivalent binding complex comprising at least two of said firstreceptor binding reagents bound to one said first multimerizationreagent; and wherein a first detectable label is bound to said firstreceptor binding reagent; and wherein a second detectable label isessentially irreversibly attached to at least one receptor molecule R.

Item 57. The cell of item 56, wherein the cell has further bound to atleast two receptor molecules R (iii) a second receptor binding reagent,the second receptor binding reagent comprising at least one secondreceptor binding site B2, wherein the second receptor binding site B2specifically binds to said receptor molecule R, the second receptorbinding reagent further comprising at least one second binding partnerC2, wherein the second binding partner C2 is capable of being stablybound to a second binding site Z2 of a second multimerization reagent,and (iv) a second multimerization reagent, the second multimerizationreagent comprising at least two second binding sites Z2 for the stablebinding of the second binding partner C2 of the second receptor bindingreagent, wherein the second receptor binding reagent (iii) and thesecond multimerization reagent (iv) form a second multivalent bindingcomplex that binds to said target cell, the second multivalent bindingcomplex comprising at least two of second receptor binding reagentsbound to one said second multimerization reagent; and wherein saidsecond detectable label is bound to said second multivalent bindingcomplex.

Item 58. The cell of item 56, wherein the cell has further bound to atleast two receptor molecules R (iii′) a multimeric receptor bindingreagent M2, the multimeric second receptor binding reagent comprising atleast two second receptor binding sites B2, wherein the second receptorbinding site B2 specifically binds to said receptor molecule R, whereinthe multimeric receptor binding reagent M2 essentially irreversiblybinds to said cell via said at least two receptor molecules R, whereinsaid second detectable label is bound to said multimeric receptorbinding reagent M2.

Item 59. The cell of item 56, wherein the cell has further bound to areceptor molecule R (iii″) an irreversible receptor binding reagent I2,wherein the irreversible receptor binding reagent I2 specifically bindsto said receptor molecule R, wherein the irreversible receptor bindingreagent I2 essentially irreversibly binds to said receptor molecule R,wherein said second detectable label is bound to said irreversiblereceptor binding reagent I2.

Item 60. The cell of any one of items 56 to 59, wherein said target cellis a mammalian cell.

Item 61. The cell of item 60, wherein said mammalian cell is alymphocyte or a stem cell.

Item 62. The cell of item 61, wherein the lymphocyte is a T cell, aT-helper cell, a B cell or a natural killer cell.

Item 63. The cell of item 62, wherein the T-cell is selected from thegroup of a CMV-specific CD8+ T-lymphocyte, a cytotoxic T-cell a, memoryT-cell and a regulatory T-cell.

Item 64. The cell of any one of items 56 to 63, wherein said receptormolecule R is a T cell receptor (TCR).

Item 65. A reagent kit for determining the dissociation rate constant(k_(off)) of a receptor molecule R on a target cell and a first receptorbinding site B1, wherein the kit comprises (i) a first receptor bindingreagent, the first receptor binding reagent comprising at least onefirst receptor binding site B1, wherein the first receptor binding siteB1 specifically binds to said receptor molecule R, the first receptorbinding reagent further comprising at least one first binding partnerC1, wherein the first binding partner C1 is capable of being reversiblybound to a first binding site Z1 of a first multimerization reagent, and(ii) a first multimerization reagent, the first multimerization reagentcomprising at least two first binding sites Z1 for the reversiblebinding of the first binding partner C1 of the first receptor bindingreagent, wherein the first receptor binding reagent (i) and the firstmultimerization reagent (ii) form a first multivalent binding complexthat binds to said target cell, the first multivalent binding complexcomprising at least two of said first receptor binding reagents bound toone said first multimerization reagent; wherein said first detectablelabel is bound or capable of binding to said first receptor bindingreagent; and (iii) a second receptor binding reagent, the secondreceptor binding reagent comprising at least one second receptor bindingsite B2, wherein the second receptor binding site B2 specifically bindsto said receptor molecule R, the second receptor binding reagent furthercomprising at least one second binding partner C2, wherein the secondbinding partner C2 is capable of being stably bound to a second bindingsite Z2 of a second multimerization reagent; and (iv) a secondmultimerization reagent, the second multimerization reagent comprisingat least two second binding sites Z2 for the stable binding of thesecond binding partner C2 of the second receptor binding reagent;wherein the second receptor binding reagent (iii) and the secondmultimerization reagent (iv) form a second multivalent binding complexthat binds to said target cell, the second multivalent binding complexcomprising at least two of second receptor binding reagents bound to onesaid second multimerization reagent; wherein a second detectable labelis bound to said second multivalent binding complex; or (iii′) amultimeric receptor binding reagent M2, the multimeric second receptorbinding reagent comprising at least two second receptor binding sitesB2, wherein the second receptor binding site B2 specifically binds tosaid receptor molecule R, wherein the multimeric receptor bindingreagent M2 essentially irreversibly binds to said target cell via saidreceptor molecule R, wherein a second detectable label is bound to saidmultimeric receptor binding reagent M2; or (iii″) an irreversiblereceptor binding reagent I2, wherein the irreversible receptor bindingreagent I2 specifically binds to said receptor molecule R, wherein theirreversible receptor binding reagent I2 essentially irreversibly bindsto said receptor molecule R, wherein a second detectable label is boundto said irreversible receptor binding reagent I2; wherein the firstdetectable label is not the second detectable label.

Item 66. The kit of item 65, further comprising a competition reagentCR, wherein the competition reagent CR is capable of competing withfirst binding partner C1 for the binding to the first binding site Z1 onthe first multimerization reagent.

Item 67. The kit of item 65, further comprising a metal chelatingreagent, wherein the metal chelating reagent is preferably of EDTA orEGTA.

Item 68. An apparatus comprising a first container containing a targetcell comprising a receptor molecule R, and (i) a first receptor bindingreagent, the first receptor binding reagent comprising at least onefirst receptor binding site B1, wherein the first receptor binding siteB1 specifically binds to said receptor molecule R, the first receptorbinding reagent further comprising at least one first binding partnerC1, wherein the first binding partner C1 is capable of being reversiblybound to a first binding site Z1 of a first multimerization reagent, and(ii) a first multimerization reagent, the first multimerization reagentcomprising at least two first binding sites Z1 for the reversiblebinding of the first binding partner C1 of the first receptor bindingreagent, wherein the first receptor binding reagent (i) and the firstmultimerization reagent (ii) are capable of forming a first multivalentbinding complex that binds to said target cell, the first multivalentbinding complex comprising at least two of said first receptor bindingreagents bound to one said first multimerization reagent; and wherein afirst detectable label is bound or capable of binding to said firstreceptor binding reagent; and wherein a second detectable label isessentially irreversibly attached to said receptor molecule R, whereinthe first detectable label is not the second detectable label; and asecond container containing a fluid comprising a) a competition reagentCR, wherein the competition reagent CR is capable of competing withfirst binding partner C1 for the binding to the first binding site Z1 onthe first multimerization reagent; or b) a metal chelating reagent,wherein the metal chelating reagent is preferably EDTA or EGTA, whereinthe first container and a second container are connected such that afluid can be transferred from the second container to the firstcontainer.

Item 69. The apparatus of item 68, wherein the first container furthercontains (iii) a second receptor binding reagent, the second receptorbinding reagent comprising at least one second receptor binding site B2,wherein the second receptor binding site B2 specifically binds to saidreceptor molecule R, the second receptor binding reagent furthercomprising at least one second binding partner C2, wherein the secondbinding partner C2 is capable of being stably bound to a second bindingsite Z2 of a second multimerization reagent, and (iv) a secondmultimerization reagent, the second multimerization reagent comprisingat least two second binding sites Z2 for the stable binding of thesecond binding partner C2 of the second receptor binding reagent,wherein the second receptor binding reagent (iii) and the secondmultimerization reagent (iv) are capable of forming a second multivalentbinding complex that binds to said target cell, the second multivalentbinding complex comprising at least two of second receptor bindingreagents bound to one said second multimerization reagent; and whereinsaid second detectable label is bound to said second multivalent bindingcomplex.

Item 70. The apparatus of item 68, wherein the first container furthercontains (iii′) a multimeric receptor binding reagent M2, the multimericsecond receptor binding reagent comprising at least two second receptorbinding sites B2, wherein the second receptor binding site B2specifically binds to said receptor molecule R, wherein the multimericreceptor binding reagent M2 essentially irreversibly binds to saidtarget cell via said receptor molecule R, wherein said second detectablelabel is bound to said multimeric receptor binding reagent M2.

Item 71. The apparatus of item 68, wherein the first container furthercontains (iii″) an irreversible receptor binding reagent I2, wherein theirreversible receptor binding reagent I2 specifically binds to saidreceptor molecule R, wherein the irreversible receptor binding reagentI2 essentially irreversibly binds to said receptor molecule R, whereinsaid second detectable label is bound to said irreversible receptorbinding reagent I2.

Item 72. The apparatus of any one of items 68 to 71 being comprised inan apparatus for flow cytometry.

Item 73. The apparatus of any one of items 68 to 72, wherein the firstcontainer is connected to the sample inlet of an apparatus for flowcytometry.

Item 74. The apparatus of any one of items 68 to 73, wherein said firstcontainer and said second container are connected via a cannula or atubing.

Item 75. The apparatus of any one of items 68 to 74, comprising a valvearranged within the connection between said first container and saidsecond container.

Item 76. The apparatus of item 75, wherein the valve is a three-wayvalve.

Item 77. A method of isolating a high-avidity T cell comprising a)determining the dissociation rate constant (k_(off)) of a T cell in asample obtained from a subject using the method according to any one ofitems 1 to 56.b) isolating said T cell from a sample obtained from saidsubject.

Item 78. The method of item 77, wherein the determined dissociation rateconstant has a value that is equal or below a given threshold value.

Item 79. The method of item 77, wherein the threshold value is in therange of about 10⁻¹ sec⁻¹ to about 10⁻³ sec⁻³.

Item 80. The method of any one of items 77 to 79, wherein the sample isa peripheral blood sample or a PBMC sample.

Item 81. The method of any one of items 77 to 80, wherein the subject isa mammal, preferably a human.

It must be noted that as used herein, the singular forms “a”, “an”, and“the”, include plural references unless the context clearly indicatesotherwise. Thus, for example, reference to “a reagent” includes one ormore of such different reagents and reference to “the method” includesreference to equivalent steps and methods known to those of ordinaryskill in the art that could be modified or substituted for the methodsdescribed herein.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the present invention.

The term “and/or” wherever used herein includes the meaning of “and”,“or” and “all or any other combination of the elements connected by saidterm”.

The term “about” or “approximately” as used herein means within 20%,preferably within 10%, and more preferably within 5% of a given value orrange. It includes, however, also the concrete number, e.g., about 20includes 20.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integer or step. Whenused herein the term “comprising” can be substituted with the term“containing” or “including” or sometimes when used herein with the term“having”.

When used herein “consisting of” excludes any element, step, oringredient not specified in the claim element. When used herein,“consisting essentially of” does not exclude materials or steps that donot materially affect the basic and novel characteristics of the claim.

In each instance herein any of the terms “comprising”, “consistingessentially of” and “consisting of” may be replaced with either of theother two terms.

It should be understood that this invention is not limited to theparticular methodology, protocols, material, reagents, and substances,etc., described herein and as such can vary. The terminology used hereinis for the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention, which is definedsolely by the claims.

All publications and patents cited throughout the text of thisspecification (including all patents, patent applications, scientificpublications, manufacturer's specifications, instructions, etc.) arehereby incorporated by reference in their entirety. Nothing herein is tobe construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention. To the extent thematerial incorporated by reference contradicts or is inconsistent withthis specification, the specification will supersede any such material.

EXAMPLES

Cells

Human CMV-specific T cell clones and murine T cell lines were generatedas described in Nauerth M, WeiBbrich B, Knall R, Franz T, Dössinger G,Bet J, Paszkiewicz P J, Pfeifer L, Bunse M, Uckert W and others.TCR-ligand k_(off) rate correlates with the protective capacity ofantigen-specific CD8+ T cells for adoptive transfer. Sci Transl Med2013; 5:192ra87-192ra87. Peripheral blood was obtained from a healthyadult donor (male). Written informed consent was obtained from thedonor, and usage of the blood samples was approved according to nationallaw by the local Institutional Review Board (Ethikkommission derMedizinischen Fakultät der Technischen Universität München) inaccordance with the Declaration of Helsinki. Whole blood was diluted 1:1with PBS (pH 7.4) and PBMCs were obtained by density gradientcentrifugation for 20 min at 2000 rpm on a layer of Ficoll.

Multimer and Antibody Staining

pMHC molecules for generation of reversible multimers carrying aTwin-Strep-Tag were refolded and multimerized as described in Nauerth etal 2013. All pMHC molecules used for the k_(off)-rate assays in thisreport were conjugated to Alexa488 maleimide fluorophore (Thermo Fisher)and multimerized Streptactin APC (IBA). Biotinylated pMHC molecules forthe generation of non-reversible multimers were refolded according tothe protocol described in Busch D H, Pilip I M, Vijh S, Pamer E G.Coordinate regulation of complex T cell populations responding tobacterial infection. Immunity 1998; 8:353-62. For multimerization, 1 μgbiotinylated pMHC molecules were incubated with 1.25 μg StreptavidinBV421 or Streptavidin PE (Biolegend) in a total volume of 50 μl for 45min. For the k_(off)-rate assay, up to 5*10⁶ cells were incubated for 45min with the reversible multimers. If indicated, CD8 antibodies (CD8eF450, eBioscience or CD8 PeCy7, Beckman Coulter) were added after 25min and incubated for additional 20 min. For live/dead discrimination,0.2 μg propidium iodide solution was added and incubated for 5 min. Cellwere subsequently washed and either used directly for the k_(off)-rateassay or additionally stained with for 10 min (unless otherwiseindicated) with the nonreversible multimer. For titration of thenon-reversible multimer, cells were either stained with 50 μl of thenon-reversible multimer (1:1), with 25 μl diluted with 25 μl FACS buffer(1:2) or with 12.5 μl of the non-reversible multimer diluted with 37.5μl FACS buffer (1:4).

FACS Analysis and Sorting

Samples were analyzed using a CyanLx 9 color flow cytometer (BeckmanCoulter). For the k_(off)-rate assay, computed parameters of theanalyzed samples were saved to enable analysis of dissociation kinetics.Cell sorting was performed on a MoFlo (Beckman Coulter). FACS data wasanalyzed with FlowJo v9.5.2 software (Tree Star, Inc.).

Flow Cytometry Based K_(off)-Rate Assay

For the performance of the k_(off)-rate assay, cells were diluted to1*10⁶-1*10⁷ cells/ml and 100 μl of the cells were added to precooledk_(off)-rate FACS tubes containing 900 μl FACS buffer. The k_(off)-rateFACS tube was mounted together with the cooling device (qutools) on theflow cytometer and analysis was initiated. After 30 s, 1 ml 2 mMD-biotin was added over the three-way valve to the cells during ongoinganalysis. Cells were analyzed for a total of 15 min. For analysis of thek_(off)-rate, fluorescence data of specific T cells were exported fromFlowJo into a spreadsheet program (GraphPad Software, San Diego, Calif.,USA). Data points for analysis of the k_(off)-rate were selected asdescribed in the text and an one-phase exponential decay curve fittedinto the data points.

Microscopic k_(off)-Rate Assay

Performed as described in detail in Nauerth et al. 2013.

Example 1: Setup and Analysis of the Flow Cytometry-Based TCR-Ligandk_(off)-Rate Assay

In order to transfer the principle of the TRC-ligand k_(off)-rate assayto flow cytometry, the basic components for data analysis need to beacquired in analogy to the original microscopy-based application: first,the initial pMHC/Streptactin multimer staining has to be determined;then, D-biotin needs to be added to the sample and subsequently thedissociation of the Streptactin backbone and the pMHCs has to befollowed until a minimum level of ligand binding is reached. Since theinventors knew already from the microscopy-based assay that for some Tcell receptors k_(off)-rate kinetics can be quite fast, they designed anexperimental setup for flow cytometry where d-biotin can be added to theprobe without interrupting the online measurement. For this purpose, theinventors penetrated standard polyethylene FACS tubes with a sharpinjection needle that was connected via a three-way valve to a syringecontaining D-biotin solution (FIG. 2A). This setting allows to acquireinitial staining values (usually 30 seconds) followed by D-biotininjection and subsequent acquisition of the dissociation phase (usually15 min) without the need to take the probe out of the sample holder atany time-point.

K_(off)-rate assays are preferably to be performed at definedtemperatures in order to obtain reproducible results that can becompared between different samples. For TCR-ligand interactions, theassay was performed at low temperatures (preferably at 4° C.). For thispurpose, the inventors designed a peltier-controlled cooling devicetightly surrounding the sample tube while still leaving a small openingfor the D-biotin injection system (FIG. 2B).

With this experimental setup, the inventors next acquired a fullk_(off)-rate sequence using a T cell line expressing the 2C TCR. Theinventors stained the cells with reversible MHC multimers based onAlexa488-labeled pMHC (H2Kb-SIYRYYGL (SEQ ID NO: 10)) and APC-conjugatedStreptactin as backbone. Additional PI staining was performed toidentify dead cells. Data analysis of the dissociation was performed bygating on the population of interest (in this case living lymphocytes)and subsequent display of the Streptactin-APC fluorescence and theMHC-Alexa488 fluorescence over the time of analysis (FIG. 2C). Tovisualize the underlying dissociation kinetics, the inventors first drew200 gates over the time of analysis and calculated the MFI ofMHCAlexa488 and Streptactin-APC for each gate. This reduction of dataallows for the plotting the MHCAlexa488 and the Streptactin-APCfluorescence values in one graph and therefore the direct comparison ofboth dissociation kinetics (FIG. 2D). At the start of the analysis, theinitial staining intensity of both fluorochromes is visible and staysconstant. Upon addition of D-biotin, a fast decay of the APCfluorescence is detectable, caused by the dissociation of the detachedStreptactin backbone. Interestingly, also the Alexa488 fluorescenceshows in the beginning a fast decay, simultaneously to theStreptactin-APC dissociation. After complete dissociation ofStreptactin-APC, a second and slower decay of Alexa488 becomesdetectable. These two kinetics of MHC-Alexa488 decay can be explained bydifferent binding states of the multimerized MHC molecules. Only a partof the MHC molecules multimerized on the Streptactin backbone is likelyto be bound to surface expressed TCRs. Several MHC molecules are onlybound to Streptactin and dissociate therefore simultaneously with thebackbone after the addition of D-biotin, showing a very fast kinetic.The following slower kinetic of Alexa488 decay results from thedissociation of monomeric TCR-bound MHC molecules from the T cellsurface and is the basis for the calculation of the TCR-ligandk_(off)-rate. These findings indicate that the k_(off)-rate can only becorrectly calculated after the complete dissociation of the non-cellbound components, as overlaying kinetics of TCR bound and unboundMHC-Alexa488 molecules are interfering. Therefore, the inventors decidedto use the time-point of complete dissociation of Streptactin-APC asstarting-point for the k_(off)-rate calculation of the MHC-Alexa488dissociation.

For the final calculation of the k_(off)-rate, the inventors directlyexported the fluorescence and time values for each analyzed cell to aspreadsheet software and generated dot plots for Streptactin-APC andMHC-Alexa488 fluorescence over the time of analysis (FIG. 2E). Theinventors then selected the data points for Streptactin-APC afteraddition of D-biotin and for MHC-Alexa488 after complete dissociation ofthe Streptactin backbone. As the exponential decay is then fitteddirectly into the dot plot of all cells, no reduction of information isperformed by calculating MFIs in a limited number of gates, which isespecially important when only limited numbers of cells can be analyzed.

In summary, the transfer of the TCR-ligand k_(off)-rate assay to theapplication on the flow cytometer allows for fast analysis of T cellsunder temperature controlled conditions and accurate calculation ofk_(off)-rates.

Example 2: The Flow Cytometry Based- and the Microscopy-GuidedTCR-Ligand k_(off)-Rate Assay Provide Comparable Values

With the microscopy-guided TCR-ligand k_(off)-rate assay, Nauert et al.2013 demonstrated a clear correlation between the k_(off)-rate and thefunctionality of analyzed T cells. Thereby, experimental setup could bestandardized the determination of highly reproducible and trulyquantitative k_(off)-rate values could be ensured. In order to testwhether results obtained from the novel flow cytometry based settingprovide comparable values, the inventors analyzed the k_(off)-rates ofCMV-specific T cell clones in parallel with both methods. Three clonesof different specificities and avidities were analyzed (FIGS. 3A-C) andin all cases the obtained k_(off)-rates were highly comparable (FIG.3D). These data demonstrate that similar robustness, reproducibility andprecision can be achieved with the novel flow cytometry based setup ascompared to the previously described microscopy-guided approach.

Example 3: Principle of Double Staining with Reversible andNon-Reversible MHC Multimers

With the experimental setup for a flow cytometry-based k_(off)-rateassay described above, analysis can only be performed with highlypurified antigen specific T cell populations, T cell clones orTCR-transduced T cells. In these cases, gating on living cells should besufficient to identify the cell population of interest throughout theacquisition of ligand dissociation kinetics. However, the analysis ofantigen-specific T cell populations directly ex vivo without previoussorting or cloning steps also requires stable identification of thetarget population during the k_(off)-rate assay. Therefore, theinventors tested whether this can be achieved by an additional antigenspecific non-reversible staining (FIG. 4A). Conventional MHC-multimersconsist of recombinant pMHCs that are biotinylated at the C-term andbind with these domains with very high affinity to Streptavidin. Thesebindings are not disrupted in significant amounts by the addition ofD-biotin, and conventional multimers therefore provide aquasi-non-reversible staining of antigen-specific T cells. FIG. 4B showsprinciple of double staining of antigen-specific T cells withnon-reversible multimers containing Streptavidin BV421 and reversiblemultimers with Alexa488 conjugated MHCs bound to a Streptactin APCbackbone. It has been described previously by others that MHC multimerdouble staining for the same specificity is generally possible, and thisapproach is now frequently used to increase the sensitivityantigen-specific T cell identification (Hadrup et al., 2009, NatMethods, 6:520-6). However, double staining with two different types ofmultimer reagents has not been tested so far. Therefore, the inventorsstained a T cell clone specific for the CMV-derived HLA-B8 restrictedepitope 1E188-96 with reversible and non-reversible specific multimers.As shown in FIG. 4B (dot plot to the top left), both multimersaccumulate with similar staining characteristics on the cell surface oflabeled cells as indicated by a directly correlating double stainingpattern. Very similar results were generated with other T cell cloneswith other epitope specificities and HLA-restrictions (data not shown),pointing towards the general feasibility of this approach. Afteraddition of D-biotin, the BV421 staining remains stable over time ofanalysis, while the MHC-Alexa488 and the Streptactin APC staining decay(FIG. 4B, bottom panels). These data show that combined staining withconventional and reversible MHC multimers represents a suitable tool toconstantly trace the cell population of interest during the TCR-ligandk_(off)-rate assay.

Example 4: Double Staining has No Influence on Reversible MultimerStaining Intensity and k_(off)-Rate

To further analyze the suitability of double staining withnon-reversible and reversible multimers for the k_(off)-rate assay inmore detail, the inventors tested if additional staining with anon-reversible reagent of the same specificity interfered withreversible multimer staining and subsequent k_(off)-rates. Therefore,the inventors stained PBMCs from a CMV+ donor with a B7pp65 specific Tcell population first with the specific reversible multimer according tothe established protocol to achieve optimal MHC-Alexa488 stainingintensity. After washing off unbound reagents, the inventors added in anadditional staining step non-reversible multimers. The inventorstitrated both concentration and incubation time over a similar initialnon-reversible multimer staining procedure. As shown in FIG. 5A and FIG.5B, increasing concentration of the non-reversible multimer onlyslightly affected the staining intensity of the non-reversible multimer(FIG. 5B). More importantly, across different concentration ofnon-reversible multimer staining, the subsequently determinedk_(off)-rates were identical under all conditions (FIG. 5C, D).Increasing the incubation time had also no detectable effects onreversible MHC multimer staining or subsequent k_(off)-rate measurements(FIG. 5D). These data demonstrate that the double staining approach withreversible and non-reversible MHC multimer reagents is robust, meaningthat slight changes in the staining conditions (incubation time,multimer concentration) do not affect subsequent k_(off)-rate results.In order to evaluate whether MHC multimer double staining could somehowaffect k_(off)-rate kinetics, the inventors compared k_(off)-ratemeasurements of T cells stained only with the reversible multimer orstained with the non-reversible multimer in addition. These experimentswere performed with well characterized high and low avidity CMV-specificT cell clones, in order to control for potential differences dependingon TCR avidity. As shown in FIG. 5E and FIG. 5F, both T cell clonesrevealed highly comparable k_(off)-rates after single or doublestaining. This clearly demonstrates that the double staining approachdid not influence resulting k_(off)-rate values and that thisobservation holds true for T cells with very different avidities.

Example 5: Double Staining Enables k_(off)-Rate Measurement of CD8+ TCell Populations Directly Ex Vivo

As the additional staining with conventional non-reversible multimers isa suitable tool to stably stain epitope-specific T cells during thek_(off)-rate assay, the inventors next investigated if the directanalysis of antigen specific T cell populations from PBMCs would bepossible with this setup. For this purpose, the inventors took bloodfrom a CMV-positive donor with a readily detectable B7pp65-specific Tcell population of 0.31% of all lymphocytes (FIG. 6A). The inventorspurified the PBMCs by density centrifugation and stained the cells withthe reversible multimer as well as a CD8 antibody, and subsequently withthe non-reversible multimer. As control, a part of the cells were onlystained with the reversible multimer and CD8 antibody, and thek_(off)-rate was analyzed after FACS sorting for Streptactin APC+ CD8+ Tcells. The gating strategy for the k_(off)-rate measurement of theB7pp65-specific T cell population directly ex vivo is shown in FIG. 6A.After gating for singlets and living lymphocytes, a gate was set forCD8+ non-reversible multimer+ T cells. This gate was used in theanalysis to display Streptactin-APC and MHC Alexa488 fluorescence overtime and to calculate k_(off)-rates. Direct export of fluorescent valuesof each measured cell was chosen by analyzing this small population toprevent loss of information. This strategy demonstrated the existence ofat least one high avidity T cell clone within the population that had aslow decay in MHC488 fluorescence over time (FIGS. 6 B, C). Thek_(off)-rates of the directly analyzed cells compared to previouslysorted cells were similar, demonstrating the reliability of directanalysis (FIG. 6D). Altogether, these data show that double stainingwith non-reversible and reversible multimers enables k_(off)-ratemeasurements of epitope-specific T cell populations directly ex vivo andprovides highly comparable results to previously sorted T cells.

Example 6: Ex Vivo k_(off) Rate Measurement of Oligoclonal HumanCMV-Specific CD8+ T Cells

Further ex vivo k_(off) rate measurement of oligoclonal humanCMV-specific CD8+ T cells from different donors were carried out asdescribed in Example 1. Here, only a fraction of the cells comprised inthe samples were CMV-specific. FIG. 7 shows ex vivo k_(off) ratemeasurement of an oligoclonal HLA*07 02/CMVpp65 specific CD8 T cellpopulation. PBMCs were isolated by Ficoll gradient centrifugation fromfresh blood of a healthy donor. Boolean gating on single living CD19−CD8+ non-reversible pMHC-PE+ T cells was performed. Data of StreptactinAPC and reversible pMHC-Alexa488 over time were exported to GraphPadPRISM to fit an exponential decay curve to determine the k_(off) rate.FIG. 8 shows Ex vivo k_(off) rate measurement of an oligo clonal HLA*0201/CMVpp65 specific CD8 T cell population (donor HZ961). k_(off) ratemeasurement was carried out on cryopreserved PBMCs isolated by Ficollgradient centrifugation from fresh blood of a healthy donor. Booleangating on single living CD19− CD8+ non-reversible pMHC-PE+ T cells wasperformed. Data of Streptactin APC and reversible pMHC-Alexa488 overtime were exported to GraphPad PRISM to fit an exponential decay curveto determine the k_(off) rate. FIG. 9: Ex vivo k_(off) rate measurementof an oligo clonal HLA*02 01/CMVpp65 specific CD8 T cell population(donor HZ510). k_(off) rate measurement was carried out on cryopreservedPBMCs isolated by Ficoll gradient centrifugation from fresh blood of ahealthy donor. Boolean gating on single living CD19− CD8+ non-reversiblepMHC-PE+ T cells was performed. Data of Streptactin APC and reversiblepMHC-Alexa488 over time were exported to GraphPad PRISM to fit anexponential decay curve to determine the k_(off) rate. These experimentsdemonstrate that k_(off) rate determination of target specific CD8+ Tcells can be carried out directly on blood or PBMC samples and thatreliable results are achieved with cells from different donors.

Example 7: Functional Characterization of Isolated TCRs Using k_(off)Rate Measurement on CD8+J76 Tumor Cells

Two HLA*02 01/CMVpp65 specific TCRs were isolated using single clonePCR. To analyze their structural avidity, TCRs were transduced intoCD8+J76 tumor cells lacking an endogenous TCR. K_(off) rate measurementwas carried out as described in Example 1. Boolean gating on singleliving CD8+ non-reversible pMHC-PE+ T cells was performed. Data ofStreptactin APC and reversible pMHC-Alexa488 over time were exported toGraphPad PRISM to fit an exponential decay curve to determine thek_(off) rate (FIG. 10). Although only a small portion of J76 tumor cellsrecombinantly expressed the respective TCR, the k_(off) values of thetwo TCRs were reliably determined. This experiment shows again that thek_(off) rate of a receptor that is only present in a small portion ofcells within a complex population can be determined using the methodsdescribed herein

Example 8: k_(off) Rate Measurement in a Murine Model System for Highand Low Avidity TCR pMHC Interaction

FIG. 11 shows k_(off) rate determination of murine T cells which wascarried out as described in Example 1. For this purpose, reversible andnon-reversible murine MHC multimers were generated according to themethod described in Example 1. The H2-KB multimers were generated usingfollowing epitopes: SIIQFEKL:H2 kb (SEQ ID NO:14), SIYNFEKL:H2 kb (SEQID NO: 15), and SIINFEKL:H2 kb (SEQ ID NO: 15). These results show thatthe methods described herein can be carried out on cells of differentspecies.

The invention illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Additionally, the terms andexpressions employed herein have been used as terms of description andnot of limitation, and there is no intention in the use of such termsand expressions of excluding any equivalents of the features shown anddescribed or portions thereof, but it is recognized that variousmodifications are possible within the scope of the invention claimed.Thus, it should be understood that although the present invention hasbeen specifically disclosed by exemplary embodiments and optionalfeatures, modification and variation of the inventions embodied thereinherein disclosed may be resorted to by those skilled in the art, andthat such modifications and variations are considered to be within thescope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the invention. This includes the genericdescription of the invention with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the invention are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

1. A method of determining the dissociation rate constant (k_(off)) ofbinding between a receptor R expressed on a surface of a target cell anda first receptor binding site B1, comprising: providing said target cellcomprising a first detectable label and a second detectable labelattached to the target cell by contacting the target cell with (i) afirst receptor binding reagent comprising said first detectable label,and at least one said first receptor binding site B1, wherein the firstreceptor binding site B1 specifically binds to said receptor R, thefirst receptor binding reagent further comprising at least one firstbinding partner C1, wherein the first binding partner C1 is capable ofbeing reversibly bound to a first binding site Z1 of a firstmultimerization reagent, (ii) said first multimerization reagentcomprising at least two said first binding sites Z1, wherein the firstreceptor binding reagent and the first multimerization reagent form afirst multivalent binding complex that reversibly attaches to receptor Ron said target cell, the first multivalent binding complex comprising atleast two of said first receptor binding reagents bound to one saidfirst multimerization reagent, and (iii) said second detectable labelthat stably attaches to said receptor R on said target cell, wherein thefirst detectable label and the second detectable label are differentfrom each other, and wherein said first detectable label and said seconddetectable label are simultaneously attached to the target cell throughdifferent receptors R; detaching the first detectable label from thetarget cell by disrupting binding between the first receptor bindingreagent and the first multimerization reagent, wherein said seconddetectable label remains stably attached to said receptor R on saidtarget cell; and identifying said target cell by detecting the seconddetectable label stably attached thereto and determining thedissociation rate constant as a function of said detaching of the firstdetectable label from the target cell determined from loss of the firstdetectable label from the identified target cells.
 2. The method ofclaim 1, wherein the cell has further been contacted with a secondreceptor binding reagent comprising at least one second receptor bindingsite B2, wherein the second receptor binding site B2 specifically bindsto said receptor R, the second receptor binding reagent furthercomprising at least one second binding partner C2, wherein the secondbinding partner C2 is capable of stably attaching to a second bindingsite Z2 of a second multimerization reagent, and said secondmultimerization reagent comprising at least two said second bindingsites Z2, wherein the second receptor binding reagent and the secondmultimerization reagent form a second multivalent binding complex thatbinds to said target cell, the second multivalent binding complexcomprising at least two of second receptor binding reagents bound to onesaid second multimerization reagent.
 3. The method of claim 1, whereinsaid second detectable label is a component of a multimeric receptorbinding reagent M2 comprising at least two second receptor binding sitesB2, wherein the second receptor binding site B2 specifically binds tosaid receptor R, wherein the multimeric receptor binding reagent M2stably attaches to said target cell via said receptor R, thereby stablyattaching said second detectable label to said receptor R on said targetcell.
 4. The method of claim 1, wherein said second detectable label isa component of an irreversible receptor binding reagent I2 thatspecifically binds to said receptor R, wherein the irreversible receptorbinding reagent I2 stably attaches to said receptor R, thereby stablyattaching said second detectable label to said receptor R on said targetcell.
 5. The method of claim 1, wherein said receptor R is a T cellreceptor (TCR).
 6. The method of claim 1, wherein said first receptorbinding site B1 is a MHC protein.
 7. The method of claim 1, wherein a.said first binding partner C1 comprises a streptavidin or avidin bindingpeptide and said first multimerization reagent comprises streptavidin,or avidin, or a streptavidin analog, or an avidin analog that reversiblybinds to said streptavidin or avidin binding peptide; or b. said firstbinding partner C1 comprises a biotin analog that reversibly binds tostreptavidin or avidin and said first multimerization reagent comprisesstreptavidin, or avidin, or a streptavidin analog, or an avidin analogthat reversibly binds to said biotin analog.
 8. The method of claim 1,comprising contacting said target cell comprising said receptor R withsaid first receptor binding reagent and said first multimerizationreagent and contacting said target cell with the second receptor bindingreagent and the second multimerization reagent, or contacting saidtarget cell with the multimeric receptor binding reagent M2; orcontacting said target cell with the irreversible receptor bindingreagent I2.
 9. The method of claim 8, further comprising (c) disruptingthe binding between the first receptor binding reagent and the firstmultimerization reagent.
 10. The method of claim 9, wherein the bindingbetween the first receptor binding reagent and the first multimerizationreagent is disrupted with a metal chelating regent.
 11. The method ofclaim 9, wherein the binding between the first receptor binding reagentand the first multimerization reagent is disrupted by pH shift.
 12. Themethod of claim 8, further comprising detecting the first detectablelabel attached to the target cell and detecting the second detectablelabel attached to the target cell.
 13. A cell comprising: receptor Rexpressed on a surface thereof, wherein the cell has bound to at leasttwo receptor R molecules (i) a first receptor binding reagent comprisinga first detectable label, and at least one first receptor binding siteB1, wherein the first receptor binding site B1 specifically binds tosaid receptor R, the first receptor binding reagent further comprisingat least one first binding partner C1, wherein the first binding partnerC1 is capable of being reversibly bound to a first binding site Z1 of afirst multimerization reagent, (ii) a first multimerization reagentcomprising at least two first binding sites Z1, wherein the firstreceptor binding reagent and the first multimerization reagent form afirst multivalent binding complex that is reversibly attached to said atleast two receptors R on said cell, the first multivalent bindingcomplex comprising at least two of said first receptor binding reagentsbound to one said first multimerization reagent, wherein the bindingbetween the first receptor binding reagent and the first multimerizationreagent can be disrupted by a metal chelating agent or by pH shift, andwherein the cell has bound to at least one receptor R molecule a seconddetectable label that is stably attached to said at least one receptor Rmolecule on said cell, wherein the first detectable label and the seconddetectable label are different from each other, and wherein said firstdetectable label and said second detectable label are simultaneouslyattached to the target cell through receptors R.
 14. The cell of claim13, wherein the cell has further bound to at least two receptor Rmolecules a second receptor binding reagent comprising at least onesecond receptor binding site B2, wherein the second receptor bindingsite B2 specifically binds to said receptor R, the second receptorbinding reagent further comprising at least one second binding partnerC2, wherein the second binding partner C2 is capable of stably attachingto a second binding site Z2 of a second multimerization reagent, andsaid second multimerization reagent comprising at least two said secondbinding sites Z2, wherein the second receptor binding reagent and thesecond multimerization reagent form a second multivalent binding complexthat binds to said target cell, the second multivalent binding complexcomprising at least two of second receptor binding reagents bound to onesaid second multimerization reagent.
 15. The cell of claim 13, whereinthe cell has further bound to at least two receptor R molecules saidsecond detectable label is a component of a multimeric receptor bindingreagent M2 comprising at least two second receptor binding sites B2,wherein the second receptor binding site B2 specifically binds to saidreceptor R, wherein the multimeric receptor binding reagent M2 stablyattaches to said target cell via said receptor R, thereby stablyattaching said second detectable label to said receptor R on said targetcell.
 16. The cell of claim 13, wherein said second detectable label isa component of an irreversible receptor binding reagent I2 thatspecifically binds to said receptor R, wherein the irreversible receptorbinding reagent I2 stably attaches to said receptor R, thereby stablyattaching said second detectable label to said receptor R on said targetcell.
 17. The cell of claim 13, wherein said receptor R is a T cellreceptor.
 18. An apparatus comprising a first container containing atarget cell comprising a receptor R expressed on a surface thereof, and(i) a first receptor binding reagent comprising a first detectablelabel, and at least one said first receptor binding site B1, wherein thefirst receptor binding site B1 specifically binds to said receptor R,the first receptor binding reagent further comprising at least one firstbinding partner C1, wherein the first binding partner C1 is capable ofbeing reversibly bound to a first binding site Z1 of a firstmultimerization reagent, (ii) said first multimerization reagentcomprising at least two said first binding sites Z1, wherein the firstreceptor binding reagent and the first multimerization reagent form afirst multivalent binding complex that reversibly attaches to receptor Ron said target cell, the first multivalent binding complex comprising atleast two of said first receptor binding reagents bound to one saidfirst multimerization reagent; and wherein a second detectable labelstably attaches to said receptor R on said target cell, wherein thefirst detectable label and the second detectable label are differentfrom each other, and wherein said first detectable label and said seconddetectable label are simultaneously attached to the target cell throughreceptors R; and a second container containing a fluid comprising a. ametal chelating agent; or b. a reagent capable of inducing a pH shift,wherein the first container and a second container are operablyconnected such that a fluid transferred from the second container to thefirst container such that the metal chelating reagent or reagent capableof inducing a pH shift disrupts binding between the first receptorbinding reagent and the first multimerization reagent, wherein saidsecond detectable label remains stably attached to said receptor R onsaid target cell.
 19. The apparatus of claim 18, wherein the firstcontainer further contains a second receptor binding reagent comprisingat least one second receptor binding site B2, wherein the secondreceptor binding site B2 specifically binds to said receptor R, thesecond receptor binding reagent further comprising at least one secondbinding partner C2, wherein the second binding partner C2 is capable ofstably attaching to a second binding site Z2 of a second multimerizationreagent, and said second multimerization reagent comprising at least twosaid second binding sites Z2, wherein the second receptor bindingreagent and the second multimerization reagent form a second multivalentbinding complex that binds to said target cell, the second multivalentbinding complex comprising at least two of second receptor bindingreagents bound to one said second multimerization reagent.
 20. A methodof isolating a high-avidity T cell comprising a) determining thedissociation rate constant (k_(off)) of a T cell in a sample obtainedfrom a subject using the method according to claim 1 to identify saidhigh-avidity T cell, wherein said receptor R is a T cell receptor (TCR),and b) isolating said T cell from the sample.